the enzymology of alanine aminotransferase (alaat) isoforms from hordeum vulgare and other...

Archives of Biochemistry and Biophysics 528 (2012) 90–101

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Archives of Biochemistry and Biophysics

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The Enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeumvulgare and other organisms, and the HvAlaAT crystal structure q

Stephen M.G. Duff ⇑, Timothy J. Rydel, Amanda L. McClerren, Wenlan Zhang 1, Jimmy Y. Li 2,Eric J. Sturman, Coralie Halls, Songyang Chen, Jiamin Zeng, Jiexin Peng, Crystal N. Kretzler,Artem EvdokimovMonsanto Company, 700 Chesterfield Parkway West, Chesterfield, MO, 63017, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 April 2012and in revised form 19 June 2012Available online 29 June 2012

Keywords:AlanineAlanine aminotransferaseCrystal structureEnzyme kineticsSubstrate specificityNitrogen metabolism

0003-9861/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.abb.2012.06.006

q Data deposition: The atomic coordinates anddeposited in the Protein Data Bank, www.pdb.org (PD⇑ Corresponding author. Fax: +1 636 737 7490.

E-mail address: [email protected] (S1 Present Address: 244 Strecker Farms Ct., Wildwoo2 Present Address: 16615 Benton Taylor Dr. Chesterfi3 Abbreviations used: AlaAT, alanine aminotransferas

AspAT, aspartate aminotransferase; BSA, bovine seanhydrase; CytC, cytochrome c; GS, glutamine synsynthase; GDH, glutamate dehydrogenase; MR, moleccrystallographic symmetry; NUE, nitrogen use efficiePGME, polyethylene glycol monomethyl ether; PLP, pyrof squares; TF HvAlaAT, tag-free barley AlaAT; Ve, elut

In this paper we describe the expression, purification, kinetics and biophysical characterization of alanineaminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzymeis a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism,and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a Km for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3,0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a kcat

of 25.6 s�1, the reverse (glutamate-forming) reaction with kcat of 12.1 s�1 and an equilibrium constantof �0.5. The enzyme is also able to utilize aspartate and oxaloacetate with �10% efficiency as comparedto the native substrates, which makes it much more specific than related bacterial/archaeal enzymes(that also have lower Km values). We have crystallized barley AlaAT in complex with PLP and L-cycloser-ine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaATstructure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga mari-tima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of Ala-AT mechanism between three kingdoms of life and allows us to shed some light on the specifics of thecatalysis performed by these proteins.

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Introduction C4 photosynthesis [2,3]. It has also been implicated in anaerobic

Alanine aminotransferase3 (AlaAT, EC 2.6.1.2) catalyzes thereversible transfer of an amino group from alanine to 2-oxoglutar-ate to form pyruvate and glutamate [1] as follows:

Pyruvateþ Glutamate$ Alanineþ 2� oxoglutarate:

It is the main route of alanine synthesis and degradation inplants and plays a pivotal role in the intercellular carbon shuttleassociated with NAD-malic enzyme- and PEP carboxykinase-type

ll rights reserved.

structure factors have beenB ID code 3TCM).

.M.G. Duff).d, MO 63011, USA.

eld, MO 63005, USA.e; AS, asparagine synthetase;rum albumin; CA, carbonicthetase; GOGAT, glutamateular replacement; NCS, non-ncy; OPA, o-phthalaldehyde;

idoxal 50-phosphate; SOS, sumion volume; V0, void volume.

glycolysis [4,5]. Given the role of AlaAT in nitrogen assimilation,protein synthesis, and carbon metabolism, it is perhaps not sur-prising that transgenic expression of AlaAT has been shown to dra-matically increase yield in rice [6] and canola [7,8]. In canola thisincrease has been linked to improvements in nitrogen use effi-ciency (NUE), as evidenced by increased nitrate influx, increasedbiomass and seed yield under low nitrogen, but not high nitrogen.

AlaAT may also mitigate a variety of stress responses in wild-type plants. Two AlaAT enzymes are induced in Arabidopsis thalianaunder hypoxic stress conditions [5]. Mutants deficient in AlaATwere shown to accumulate more Ala during the hypoxic stress per-iod and during the subsequent recovery period following stress re-moval. This suggests that under these conditions, AlaAT is moreinvolved in the breakdown of Ala rather than the synthesis, in con-trast to its role in NUE. However in maize, AlaAT expression is in-duced by both hypoxic and nitrogen stress [9], highlighting theimportance of the reversibility of the AlaAT reaction. The role ofAlaAT in plant drought tolerance has also been examined. AlthoughAlaAT activity did not increase during the onset of drought stress,there was a slight increase in the activity upon rehydration [10].

The role of AlaAT in both N uptake and stress tolerance is notcompletely understood; however, the high yield phenotype of

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S.M.G. Duff et al. / Archives of Biochemistry and Biophysics 528 (2012) 90–101 91

AlaAT in crop plants may depend on several factors including thephysical or kinetic properties of the enzyme. AlaAT catalyzes areversible reaction with an equilibrium constant near one,indicating that the reaction has no real preference towards eitherGlu or Ala formation. Although the direction of the reaction de-pends on the concentrations of each of the four substratesin vivo, it is possible that the yield phenotype could be enhancedwith an AlaAT enzyme that has specific or improved kinetic prop-erties. The yield phenotype could also be impacted by the substratespecificity of the AlaAT enzyme, since the reaction or reactions per-formed by the enzyme in vivo will direct its effect on metabolism.

In order to find a suitable AlaAT candidate for transgenic stud-ies, a comparison of the kinetic properties of several recombinantAlaAT forms was performed. The maximal velocity or catalytic effi-ciency (kcat) of each enzyme and the binding constants (Km) for thesubstrates in both directions were determined. We have also care-fully examined the substrate specificity of barley AlaAT against anexhaustive panel of potential natural substrates and shown it tohighly favor alanine. Using these results, we have developed amethod to test substrate specificity by aspartate aminotransferaseactivity, which can be used to quickly indicate how broad the sub-strate specificity of an aminotransferase is likely to be. We haveinvestigated the subunit structure of recombinant barley AlaATand some other properties, including sensitivity to inhibitors, anddependence upon the cofactor, PLP. All of this information will beuseful to determine the properties of the enzyme most necessaryfor producing a positive phenotype in crops. Finally, we have deter-mined the X-ray crystal structure of an N-terminally His-taggedform of barley AlaAT to 2.7 Å resolution. This represents the firstplant AlaAT enzyme structure to be solved.

Methods

Cloning, expression and purification of AlaAT enzymes

Table 1 contains a list of AlaAT genes from different organismsused in this study. In order to analyze the characteristics of theseenzymes, the genes encoding them were identified from eithercDNA libraries or Monsanto plant expression vectors and amplifiedby PCR. The Hordeum vulgare (Barley) gene was isolated using RT-PCR from barley seedling RNA. Using DNA polymerase in the pres-ence of dNTP, sticky ends were produced and the gene was in-serted into an appropriate pET vector, which would allow for theproduction of the enzyme with a six residue histidine tag at theN-terminus. A construct for the production in Escherichia coli oftag-free barley AlaAT (His6-SUMO-Hv.AlaAT, [11]) was made usinga similar method. The vectors were then transformed into DH5acells and confirmed by sequencing and restriction digest.

The vectors were transformed into BL21DE3 E. coli cells forexpression. For His-tagged enzymes, cells were grown in flaskscontaining LB media (400 mL) and induced by 1 mM IPTG. Afterinduction (4 h) cells were collected by centrifugation and storedat �80 �C. For the expression of the tag-free form (His6-SUMO-Hv.AlaAT), a 2 mL culture was grown in LBG media at 25 �C for15 h and then transferred into a 2 L flask containing 500 mL

Table 1AlaAT isoforms forms tested in this report.

AlaAT homologue Identifier

Hordeum vulgare AlaAT emb|CAA81231.1Saccharomyces cerevisiae AlaAT gi|1360460Oryza sativa AlaAT gi|4730884Zea mays AlaAT 1 gi|39935662Zea mays AlaAT 2 gi|21213577Bacillus halodurans D-AlaAT gi|15615374Rhodopseudomonas palustris D-AlaAT gi|39935662Pseudomonas putida b-AlaAT gi|24981984

auto-induction media [12] with antibiotics. Cells were grown at37 �C for 4 h then transferred to 25 �C overnight, then harvestedand stored at �80 �C.

For purification of the his-tagged enzymes, bacterial pelletswere resuspended in 5 mL/gfw AlaAT extraction buffer (100 mMTris, pH 8.0, 10 mM DTT, and 15% (w/v) glycerol). This suspensionwas passed through a French Press twice at 16,000 PSI, and thencentrifuged at 75,000g for one hour. The supernatant was loadedonto a 5 mL nickel NTA agarose column that was preequilibratedwith AlaAT extraction buffer. The column was washed with thesame buffer until the OD280 of the eluate was lower than 0.05.The protein was then eluted with the extraction buffer containing500 mM imidazole and desalted on a small G-25 column equili-brated with AlaAT extraction buffer.

To purify the tag-free barley AlaAT (TF HvAlaAT), a 1 L cell cul-ture pellet was resuspended in 50 mM Tris, pH 8, 2 mM b-mercap-toethanol, 0.5 tablet of EDTA-free protease (Roche), 2 mM MgCl2,and benzonase (Novagen Inc., Madison, WI, USA). Cells were lysedby two passages through the French press at 16,000 psi. The result-ing lysate was clarified by centrifugation at 20,000g and the super-natant loaded onto a 20 mL Ni2+-NTA FF column pre-equilibratedin 50 mM Tris, pH 8, 0.5 M NaCl, 20 mM imidazole, and 2 mM b-mercaptoethanol. A linear gradient was applied with the same buf-fer containing 0.5 M imidazole. A single elution peak was observedby monitoring the UV absorption at 280 nm and the correspondingfractions were pooled and concentrated for desalting via a 20 mLGM-25 column. The desalted sample was incubated overnight withSUMO protease at 4 �C with gentle inversion (�10 units SUMO/1 mg AlaAT). Alternatively, the sample was incubated with SUMOprotease for two hours at 30 �C. Digestion products were resolvedby immobilized metal affinity chromatography using the same Ni2+

column described above. Tag-free Barley AlaAT was recovered fromthe flow-through, while undigested HvAlaAT and His6-taggedSUMO were retained on the column. Following concentration anddesalting, the tag-free enzyme was applied to a 10 mL MonoQ.Tag-free HvAlaAT was eluted from the Mono-Q with a linear gradi-ent of 0 to 1 M NaCl and the UV absorption at 280 nm was moni-tored. Several peaks were observed and those containing TFHvAlaAT were pooled following SDS–PAGE to confirm the identityand purity of the sample. Glycerol was added to the final sample sothat the buffer contained 50 mM Tris, pH 8, 200 mM NaCl, 10%glycerol, and 2 mM b-mercaptoethanol. The final protein concen-tration from the MonoQ fractions ranged from 9–13 mg/mL, yield-ing �60 mg of protein per liter of cell culture.

For crystallographic studies of HvAlaAT, amino acids with highentropy side chains (lysine, glutamine and glutamate) were identi-fied and mutated to alanine or histidine similar to what has beenpreviously described by Derewenda [13] to generate 18 mutants(Table 2). Mutants were generated via QuikChange� procedures(Agilent Technologies, Santa Clara, CA, USA). Plasmids were puri-fied and confirmed by sequencing.

Mutated plasmids were transformed into BL21 DE3 E. coli cellsfor evaluation of expression and protein purification. For expres-sion (His10 �HvAlaAT), a 2 mL culture was grown in LB media at25 �C for 15 h and then transferred into a 2 L flask containing500 mL auto-induction media [12] with antibiotics. Cells weregrown at 37 �C for 4 h then 15 �C for 48 h and then harvested bycentrifugation and stored at �80 �C prior to protein purification.

For protein purification, cell pellets from each 500 mL culturewere suspended in 125 ml of 20 mM Tris pH 8, 150 mM NaCl,2 mM beta mercaptoethanol, 2 mM MgCl2, 1 � Roche complete pro-tease inhibitor cocktail without EDTA, 5 U/mL benzonase nuclease(Novagen Inc., Madison, WI, USA) and 5000 U/mL lysozyme (RocheApplied Science, Indianapolis, IN, USA), and were mechanically dis-rupted with a cell disruptor (Constant cell disruption systems, mod-el TS5) at 20 PSI for one pass. Lysates were cleared with a 35,000g

Table 2Surface-entropy reduction variants for enhanced crystallization.

Mutant portion of mutations (nudeatides) AA mutations

Nl 37AA/GC, 47AA/GC K13A, K16SN2 88CA/GC, 97CAG/AGC, 1O3CA/GC, 109 A/

C, lllG/T, and 115CAA/GCQ30A, Q33S, Q35A,K37H, and Q39A

N3 169CAA/GCG Q57AN4 355A/Cand 357A/T K119HN5 469CAA/AGC Q157SN6 623AGCAA/GCGCG K208Sand Q209AN7 712GAA/AGC and 721CAA/GCG E238SandQ241AN8 736AA/GC and 745AA/GC K246Aand K249AN9 883CAA/GCG and 895AA/GC Q295Aand K299ANlO 938A/C E313ANll 967GAGCA/AGCGC E323S, Q324A,

and 979AAA/GCG and K327ANl 1018CA/GC Q340ANl3 1064AA/CG E355ANl4 1126AA/GC K376ANl5 1127AG/GC, 1136A/C, and 1150AAA/GCG K376S, E379A, and

K384ANl6 1289A/C E430ANl7 1381AA/GC K461ANl8 1436A/C E477A

92 S.M.G. Duff et al. / Archives of Biochemistry and Biophysics 528 (2012) 90–101

centrifugation for 30 min followed by 0.2 lm filtration. Lysates wereloaded onto a 5 ml HisTrap FF (GE Healthcare, Little Chalfont, UK)column and peak fractions were loaded onto a HiLoad 16/60 Super-dex 30 desalting column (GE Healthcare, Little Chalfont, UK). De-salted samples were further purified on a 5/50 GL MonoQ (GEHealthcare, Little Chalfont, UK) column with a 20 column volumegradient from 0–1 M NaCl. Peak fractions (as visualized on SDS–PAGE) were pooled and concentrated with a Millipore AmiconUltra-4 Ultracel 10 K. Concentrated samples were used for sizeexclusion chromatography on a Superdex 200 HiLoad 16/60 prepgrade column (GE Healthcare, Little Chalfont, UK) with 20 mM TrispH 7.8, 150 mM NaCl and 2 mM beta mercaptoethanol. For initialcrystallization experiments, peak fractions were pooled, concen-trated, and buffer exchanged using a Millipore Amicon Ultra-4 Ultra-cel 10 K into the final buffer of 20 mM Tris buffer pH 8.0, 150 mMNaCl, 2 mM beta mercaptoethanol.

AlaAT coupled activity assay

Routine and kinetic measurements of AlaAT were performedusing a coupled enzyme assay in both reaction directions. In theglutamate-forming direction AlaAT activity was coupled to that oflactate dehydrogenase and assayed at 25 �C by monitoring NADHutilization at 340 nm using a 96-well plate reading spectrophotom-eter. Model standard assay conditions were: 100 mM Hepes (pH8.0), 0.1 mM NADH, 5 U/mL lactate dehydrogenase, 10 mM alanine,and 2 mM 2-oxoglutarate where one unit of AlaAT activity was de-fined as the amount of enzyme which converts 1 lmol NADH/min.Values are presented in specific activity (lmol/min/mg protein) un-less otherwise indicated and typical assay volumes were 0.2 mL.The assay was initiated by the addition of 2-oxoglutarate. In the ala-nine-forming direction AlaAT activity was coupled to glutamatedehydrogenase and assayed at 25 �C by monitoring NADH utiliza-tion at 340 nm using a 96-well plate reading spectrophotometer.Standard assay conditions were 100 mM Hepes (pH 8.0), 0.1 mMNADH, 6 U/mL glutamate dehydrogenase, 2 mM EDTA, 100 mMNH4Cl, 25 mM glutamate, and 10 mM pyruvate. Assays were con-ducted in the presence or absence of pyridoxal 50-phosphate (PLP)as indicated. The assay was initiated by the addition of pyruvate.

Kinetic Analysis of AlaAT

The kinetic parameters measured were Km (ala), Km (glu), Km (pyru-vate), Km (2-oxoglutarate) and kcat, for all substrates and in both direc-

tions. kcat/Km and Hill coefficients (h) were also determined for eachsubstrate. Additionally, the I0.5 (concentration of inhibitor at whichthe enzyme maintains 50% of its maximal activity) was also deter-mined for several putative inhibitors with the tag-free barley AlaAT.

Apparent Km for each substrate and Vmax values were evaluatedfrom Lineweaver–Burk and Hill plots, respectively [14]. kcat was esti-mated from Vmax using the derived molecular mass of each enzyme.Hill coefficients (h) were determined from the latter. Km values foreach substrate were determined at a standard concentration(10 mM amino acid or 2 mM keto acid) of the other substrate. In addi-tion substrate titrations of Glu and Ala were made at different con-centrations of pyruvate and 2-oxoglutarate, respectively, toinvestigate the reaction order. The concentration range of substratesused to determine the Km values varied depending upon the substrateand isoform, however, at least 9 separate substrate concentrationswith at least 4 concentrations above and 4 below the final Km valuewere used. The I0.5 (inhibitor) values were determined from a Job plot[15] using the concentration of substrate and inhibitors described inthe Results. All rates determined from activities in which substrateconcentrations were above saturating were omitted from the Km orI0.5 determinations. All kinetic parameters are the means of duplicatedeterminations performed on at least two separate preparations ofeach purified enzyme (i.e., n = 4), and are reproducible to within±15% SE (or better). All kinetic plots were constructed and constantsdetermined using GraFit Version 5 (Erithacus Software, Surrey, UK)with the exception of the double reciprocal plot in Fig. 3.

The equilibrium constants for the reaction catalyzed by barleyAlaAT were calculated from our kinetic values using the Haldaneequation (Haldane 1930) and compared to values from a public data-base (NIST standard reference database 74 for thermodynamics ofenzyme catalyzed reactions, http://xpdb.nist.gov/enzyme_thermo-dynamics/).

Aminotransferase HPLC activity assay

In order to measure substrate specificity, an aliquot of tag-freebarley AlaAT was incubated with potential substrates. Since theoverall level of activity varies greatly between substrates, theamount of AlaAT and the time of incubation were determinedempirically, although assays were configured such that 10–15 minincubation put the reactions in the linear range. Substrate concen-trations used were 25 mM amino acid (donor) and 10 mM keto acid(acceptor). These concentrations are considerably above kcat levelsfor tag-free barley AlaAT with the standard substrates (Glu, Ala, 2-oxoglutarate, pyruvate) in order to detect possible transaminationreactions which were considerably less efficient than glutamate toalanine. The assay was stopped by adding trichloroacetic acid to a fi-nal concentration of 10% (v/v). The reaction mixture was then clari-fied by filtration or centrifugation and an amount of 0.5 ll ofsupernatant was analyzed for free amino acids. The amount of theexpected product amino acid was determined and used to calculatethe activity of AlaAT and the results were calculated as percent activ-ity compared to the best substrates (glutamate and pyruvate).

The HPLC system consisted of an Agilent 1100 HPLC with a cooledautosampler, a fluorescence detector, and a HP Chemstation data sys-tem. Separation of the amino acids was performed using precolumno-phthalaldehyde (OPA) derivatization followed by separation byHPLC using a Zorbax Eclipse-AAA 4.6� 75 mm, 3.5 lm column.Detection was by fluorescence (excitation wavelength was 235 nm),and chromatograms were collected using the HP Chemstation. Allstandards and reagents were purchased from Agilent Technologies.

Coupled activity assay for AspAT

For enzymes other than tag-free barley AlaAT, aspartate amino-transferase activity was measured and used as a general indicatorof substrate specificity.




The activity was measured in the aspartate-forming direction in amanner very similar to the AlaAT assay. The activity was coupled withglutamate dehydrogenase and assayed at 25 �C by monitoring NADHutilization at 340 nm using a 96-well plate reading spectrophotome-ter. Standard assay conditions were 100 mM Hepes (pH 8.0), 0.1 mMNADH, 6 U/mL glutamate dehydrogenase, 2 mM EDTA, 100 mMNH4Cl, 25 mM glutamate, 10 mM oxaloacetate. Assays were con-ducted in the presence of PLP as indicated, where 1 unit of AspATactivity is defined as the amount of enzyme which can convert 1 lmolNADH/min. The assay was initiated by the addition of oxaloacetate.

Analysis of barley AlaAT structure by analytical gel filtration

In order to determine the oligomerization state of HvAlaAT,analytical gel filtration was performed using a 24 mL Superdex200 column. The nominal resolving capacity of this column rangesfrom 3000–600,000 Da. The column was equilibrated with 50 mMTris, pH 8, 200 mM NaCl, 1 mM DTT at 0.5 mL/min for severalhours. Two column volumes of buffer were passed over the columnin a single run at the same rate in isocratic mode. The void volume(V0) of the column was first determined by injecting a 1 mg/mLsample of Blue Dextran (MW = 2,000,000) onto the column. A mix-ture of protein standards was next resolved in order to develop astandard mass curve. The protein standard mixture was preparedin the column run buffer as follows: alcohol dehydrogenase was4.4 mg/mL (ADH), bovine serum albumin was 7.8 mg/mL (BSA),carbonic anhydrase was 2.2 mg/mL (CA), and cytochrome c was1.6 mg/mL (CytC). The absorbance at 280 nm was monitored anda single elution peak for each protein was observed. Finally, a sam-ple of HvAlaAT was desalted into the column running buffer andinjected onto the column at �2 mg/mL, where a single elution peakwas also observed. Injection volumes were kept constant for allthree runs. By measuring the elution volume (Ve) for each of thestandards and HvAlaAT, the Kav for each protein could be deter-mined by the following equation:

Kav ¼ ðVe � V0Þ=ðVt � V0Þ;

where Ve is experimentally determined for each protein, V0 = 8 mL,and Vt = 21.84 mL.

Determination of the PLP-binding of Barley AlaAT

A tag-free enzyme preparation of HvAlaAT was prepared at4.5 mg/mL in 50 mM Tris, pH 8. The UV–visible spectra were exam-ined from 300 to 500 nm and a maximum at �420 nm was ob-served. This recombinant protein sample was then treated with5 mM hydroxylamine in 50 mM Tris, pH 8 and the spectra werereexamined across the same range, where a shift in absorbancemaxima from 420 to �380 nm was observed, attributable to theformation of a PLP oxime.

Additive/buffer screen anddynamic light scattering for crystallography

Three HvAlaAT enzymes, specifically the native enzyme, and vari-ants N2 (Q30A, Q33S, Q35A, K37H, and Q39A) and N4 (K119H) fromTable 2, were subjected to an additive/buffer screen and evaluatedby dynamic light scattering. Buffer pH’s were adjusted to 5.0(20 mM NaAcetate), 7.8 (20 mM Tris–HCl) and 10.0 (20 mM SodiumCarbonate). Additives included for each pH were 2.5 mM DTT (reduc-ing agent), 0.1 M guanidine HCl (chaotrope), 3% glycerol (polyol),100 mM glycine (linker), 2.5 mM PLP (co-factor) or 2.5 mM L-cycloser-ine (inhibitor). Protein buffer was exchanged for the new additive buf-fer by loading 100 lL of the purified protein sample on a PD MultiTrapG-25 plate (GE Healthcare) and centrifugation as recommended bythe manufacturer. DLS experiments were carried out using a WyattDynaPro Plate Reader (Wyatt Technologies, Santa Barbara, CA). Sam-ples were placed in a 354-well Corning plate model 3540 (Corning,

NY). For each sample, 20 lL was placed in the plate followed by5 lL of paraffin oil to avoid evaporation, and each plate was spun at1000g for 10 s to remove trapped air bubbles. Ten data acquisitionsof 10 s each were scheduled and collected by the instrument software,followed by fitting of the autocorrelation function. Filters were set foran amplitude minimum of 0 and a maximum of 1. The baseline wasset to 1 ± 0.01 and a maximum sum of squares (SOS) of 100. Any datathat fell outside of accepted ranges were culled. DLS plates werestored at 4 �C for 3 days and measured again in the same manner.

Crystallization, X-ray data collection, structure determination, andrefinement of HvAlaAT-K119H

Initial vapor diffusion crystallization experiments were con-ducted on the native HvAlaAT enzyme and all variants listed in Ta-ble 2. Further crystallization work was performed using the threeprotein samples deriving from the additive and DLS screen.Numerous crystallization conditions were tested by using a varietyof commercially-available reagent screen kits. The HvAlaAT-K119Henzyme was successfully crystallized by vapor diffusion in sittingdrops using Cryschem plates at room temperature. The proteinsample was 12 mg/mL in 20 mM carbonate buffer-pH 10.5,2.5 mM PLP, 2.5 mM cycloserine, and 2.5 mM DTT. Crystals wereobtained using a 0.7 mL well composition of 22 (w/v)% polyethyl-ene glycol monomethyl ether (PGME) 5000, 0.1 M MES-pH 6.0 buf-fer, 9 (v/v)% tacsimate-pH 6.0 and a 2 lL sitting drop prepared fromequal volumes of protein and well solution.

The HvAlaAT-K119H crystals were prepared for low tempera-ture X-ray data collection by dipping them briefly in a cryosolution(33% PGME 5000 and 0.1 M MES-pH 6.0 buffer) prior to plungingthem into liquid nitrogen. X-ray diffraction data were collectedon these crystals at the Southeast Regional Collaborative AccessTeam (SER-CAT) 22-ID beamline at the Advanced Photon Source,Argonne National Laboratory. The dataset used for structure deter-mination was collected from a cryocooled crystal at 100 K, and at awavelength of 1.0 Å, using a Marresearch mar300 CCD detector.One hundred and thirty degrees of data were collected using 5 sexposures, an oscillation angle of 1.0�, and a crystal-to-detectordistance of 250 mm. These data were processed using both theHKL2000 [16] and d�trek [17] programs.

An initial phasing solution for HvAlaAT was obtained by molec-ular replacement (MR). Programs from the CCP4 (CollaborativeComputational Project, Number 4) software suite for macromolec-ular crystallography, as implemented in the ccp4i graphical userinterface [18], were employed to reduce data and perform MR.Phaser [19] was used to obtain the initial MR solution from thed�trek-processed X-ray intensity data to 2.7 Å resolution; thephasing template was prepared by the program chainsaw [20]from the Pyrococcus furiosis Pfu-1397077-001 (PDB entry 1xi9)coordinate file and the HvAlaAT-PfAlaAT sequence alignment.The program RESOLVE [21] was used to perform density improve-ment with averaging. Crystallographic refinement, employing non-crystallographic symmetry (NCS) restraints, was conducted usingthe program refmac5 [22] and the HKL2000-processed data.Map-fitting and model-building were carried out using the Cootprogram [23]. Structurally bound water molecules and ligandswere identified and included in refinement based on differenceelectron density map and bonding interaction criteria.

Results

Kinetic comparison of AlaAT forms

The degree of identity between various amino acid sequences ofAlaAT investigated in this study appears in Fig. 1. Barley AlaAT wasnearly identical to maize AlaAT-1 (90% + sequence identity).

Fig. 1. Amino acid identity of alanine aminotransferase proteins described in this study. AlaAT genes are identified in Table 1.

Table 3kcat and Km values of AlaAT from various species of plant, yeast, and bacteria. Thegenes from which the enzymes were derived are described in Table 1. Values areaverages of 2 determinations on at least 2 separate enzyme preparations (n = 4). Unitsfor Km is are mM; units for kcat are s�1. Standard errors are given in parentheses.

Km

(ala)Km

(2-oxo)kcat

(glu forming)Km

(glu)Km

(pyr)kcat

(ala forming)

Sc AlaAT 3.60 0.20 6.09 0.24 0.40 8.64HvAlaAT 0.83 0.18 8.27 0.54 0.21 9.09Tag-free HvAlaAT 3.79 0.28 12.11 0.79 0.17 25.62Os AlaAT 0.40 0.25 4.98 0.68 0.11 6.86Zm AlaAT 1 0.62 0.37 5.49 0.56 0.42 6.56Zm AlaAT 2 3.06 0.20 0.33 0.55 0.54 0.67Bh D-AlaAT 5.15 0.32 1.25 0.44 0.08 2.80Rp D-AlaAT 1.52 0.20 0.38 0.20 0.08 3.06Pp b-AlaAT 0.21 0.05 0.08 0.57 0.20 0.17


The kcat and Km values were determined for this same set ofpurified recombinant AlaATs from plants and bacteria for each ofthe four substrates and are shown in Table 3. Two maize, one rice,and one barley gene were chosen to represent the plant enzymes.Three bacterial species (Pseudomonas putida, Bacillus halodurans,and Rhodopseudomonas palustris) were also included. The kineticvalues in Table 3 were determined for each enzyme in the presenceof saturating PLP (200 lM). kcat values for glutamate formationranged from 0.17 to 24.5 s�1 and were generally slightly higherthan kcat values for alanine formation which ranged from 0.08 to12.1 s�1. The kcat values for both plant and bacterial AlaAT enzymessurveyed in this report are generally low compared to rat liver Ala-AT which has a reported kcat of 17.1 s�1 for glutamate formation[24] and Pyrococcus furiosus AlaAT which has a reported kcat of142 s�1 for alanine formation [25] at 80 �C. Since over expressionof barley AlaAT has been shown to increase yield in crop plants[6] we tested both a tag-free and N-terminal his-tagged version.Tag-free (TF) barley alanine aminotransferase (HvAlaAT) had arelatively high kcat in both the glutamate and alanine formingdirections (24.5 and 12.1 s�1, respectively) compared to the otherenzymes studied. kcat values of the tag-free enzyme were consider-ably higher in both directions than those of its his-tagged form, aresult potentially due to slight differences in the purification strat-egy for the two forms [26].

Substrate Km values of the AlaAT enzymes surveyed varied overtwo orders of magnitude from 0.05 to 5.2 mM (Table 3). Hill coef-ficients were determined for all of the enzymes (for each substrate)to be between 0.9 and 1.1, suggesting that there is no cooperativitywith respect to substrate binding. Some patterns in the kinetic datacould be observed. Km values (0.05 to 0.54 mM) for the aminoacceptors (keto acids) were almost always lower than the Km

values (0.2 to 5.2 mM) for the amino donors (amino acids), sug-gesting that these enzymes normally encounter levels of their ami-no acid substrates much higher than their organic acid substrates.In addition, Km values were often but not always lower for the sub-strate of the alanine-forming reaction, suggesting that the main

function of many of these enzymes is to produce alanine. However,the Km values reported here are still generally higher than the low-est reported Km value for an alanine aminotransferase, that fromAtriplex spongiosa which has a Km (2-oxoglutarate) of 0.03 mMand Km (pyruvate) of 0.02 mM [27]. Interestingly, the Km (Ala) va-lue for the N-terminal tagged barley enzyme is almost an order ofmagnitude less than that of the tag-free version, suggesting thatthe tag may have a ‘sticky’ property that helps the enzyme bindAla more tightly.

Km and kcat values were also determined for the tag-free HvA-laAT with all substrates in the presence versus absence of exoge-nous PLP. The double reciprocal plot for the substrate alanine isshown as an example (Fig. 2). PLP had very little effect (less than5%) on Km values of tag-free HvAlaAT, while the kcat values wereup to 2-fold higher in the presence of PLP (data not shown). Acti-vation of several other enzymes was also tested. 20–30% higher en-zyme activity was observed in the presence compared to theabsence of PLP for these His-tagged enzymes at saturating sub-strate concentrations. These results indicate that AlaAT enzymesusually co-purify with their PLP cofactor bound and that PLP playsan important role in the chemistry of the reaction, since the addi-tion of PLP affects kcat rather than Km values.

Reaction mechanism of barley AlaAT

A double reciprocal plot of alanine vs. velocity at several con-centrations of 2-oxoglutarate is shown in Fig. 3. Parallel lines indi-cate the reaction mechanism is bi–bi ping–pong and that eachsubstrate binds sequentially and competitively to the enzyme asobserved for other alanine and aspartate aminotransferases [25].

Substrate specificity of AlaAT enzymes

Many bacterial aminotransferases including those annotated asAlaAT or AspAT have a broad range of either amino donors or aminoacceptors [28]. Substrate specificity of only a few eukaryotic AlaATshas been examined, and the results suggest that these enzymesmay have a narrower substrate suite [24,29,30]. We were interestedin determining the substrate specificity of the enzymes described inTable 1, and especially HvAlaAT, since the enzyme’s ability to cata-lyze more than one reaction could alter the proposed mode of ac-tion of the yield phenotype in canola [8]. A detailed substrateprofile was determined for the tag-free barley AlaAT (Table 4) at10 mM amino acceptor (keto acid) and 25 mM (amino acid). Atthese levels of the standard substrates, variable substrate and prod-uct inhibition typically occur [28]. However, since the enzyme usesalternative substrates very inefficiently, high levels were requiredto observe activity. Activity was monitored by HPLC and then con-firmed by a coupled assay whenever such an assay was available forthe substrate pair. By far the best activity demonstrated by HvAlaATwas observed for the interconversion of glutamate and pyruvate toalanine and a-ketoglutarate (AlaAT). There was some activity for

Fig. 2. Substrate titration and double reciprocal plot of the titration of alanineagainst activity for tag free barley AlaAT with (A) 200 lM PLP, (B) 0 lM PLP. Thedouble reciprocal plot is shown in the inset.


the interconversion of glutamate and oxaloacetate with aspartateand a-ketoglutarate (AspAT activity) but it was less than 10% ofthe activity of AlaAT under the assay conditions. No other substrate

Fig. 3. Double reciprocal plot of 1/alanine vs. 1/v at several concen

pairs were converted at levels within the detection limit of the as-say. Neither D-alanine nor b-alanine could be converted by the en-zyme when used as substrates in this test.

The possibility of competition between alanine and aspartatewas also examined. When the reaction was measured throughpyruvate formation (from alanine) at 2.5 mM Ala and 10 mM Aspno inhibition by Asp was observed suggesting that aspartate bindsonly very weakly to the enzyme and is unlikely to be a substratein vivo.

In order to determine the capacity for other AlaAT enzymes touse additional substrates, each protein was assayed for AspATactivity using a coupled assay. Since the only other substantialreaction catalyzed by HvAlaAT was AspAT, this reaction was cho-sen as a general indicator of substrate specificity. All tested plantAlaAT forms had AspAT activity between 5% and 10% of AlaAT sug-gesting that they are probably intended to be specific for Ala pro-duction or utilization in vivo (Table 5). The level of substratespecificity observed in this test is comparable to that describedfor other eukaryotic enzymes [24,29–32]. Bacterial enzymes testedin this study were much less specific than the plant ones, with Ala-AT and AspAT activity levels within �15% of each other (Table 5).This result indicates that they probably have fairly broad substratespecificity, especially considering their annotation as D- or b-ami-notransferases which is consistent with literature reports [28]. Oneexample of an AlaAT with an intermediate specificity is that from P.furiosus, which is reported to be less specific than the plant en-zymes described here but more specific than the other prokaryoticenzymes, perhaps reflecting its hypothetic function along with glu-tamate dehydrogenase to form an electron sink in the archeon [25].

Inhibitor effects on barley AlaAT

We investigated the effects of known aminotransferase inhibi-tors on the activity of this enzyme, including O-carboxymethylhydroxylamine hemihydrochloride, DL-vinylglycine, DL-propargyl-glycine, 4-chloromercuribenzoic acid, cycloserine, penicillamine.Both O-carboxymethyl hydroxylamine hemihydrochloride andpenicillamine are more general inhibitors of pyridoxal 50-phosphatecontaining enzymes, since they react with the pyridoxal moiety toform an oxime [33,34]. Cycloserine is a structural analog of alanine,and is known to be a more effective inhibitor of AlaAT activity thanother transaminases [33]. Inactivation by cycloserine is irreversible

trations of 2-oxoglutarate (2 mM, 0.7 mM, 0.3 mM, 0.2 mM).

Table 4Activity of Tag-Free Barley AlaAT using various amino donors and acceptors. Percentactivity is relative to the activity of the enzyme with glutamate and pyruvate as asubstrate, specifically for the HPLC and coupled assay, respectively. Results are theaverage of duplicate assays repeated on 2–4 separate preparations of tag-free barleyAlaAT. nd = not determined. Standard errors are given in parentheses.

Substrate 1 Substrate 2 HPLC assayactivity

Coupled assayactivity

Glutamate Pyruvate 100 (8) 100 (10)Alanine a-Ketoglutarate 33 (2) 28 (3)Glutamate Oxaloacetate 3 ndAspartate a-Ketoglutarate 7 3Glycine Pyruvate <1 ndAlanine Glyoxylate <1 ndGlutamate Succinate

semialdehyde0 nd

Glycine Oxaloacetate 0 ndGlycine a-Ketoglutarate 0 ndSerine Pyruvate 0 ndGlutamate Pyruvate 0 ndGABA a-Ketoglutarate 0 ndAspartate Glyoxylate 0 ndAlanine Hydroxypyruvate 0 nd

D-Alanine a-Ketoglutarate 0 nd

Alanine Oxaloacetate 0 ndAlanine Phenylpyruvate 0 ndGlutamate Glyoxylate 0 ndPhenylalanine Pyruvate 0 ndb-Alanine Pyruvate 0 ndNH4 Pyruvate 0 nd

Table 6The effect of known aminotransferase inhibitors on the activity barley AlaAT. AlaATwas assayed in the pyruvate-forming direction by the coupled assay using 0.4 mM alaand 0.2 mM a-ketoglutarate. From left to right the bars represent control with noinhibitor added, and then samples with 25 lM O-CarboxyMethyl hydroxylaminehemihydrochloride, DL-vinylglycine, DL-propargylglycine, 4-chloromercuribenzoicacid, cycloserine, and penicillamine. The results are described as the average specificactivity of two determinations on two separate preparations of the enzyme (n = 4).Inhibition of the enzyme was similar regardless of the direction of the enzyme.

Inhibitor Specific activity(U/mg)

Standarderror

Control 2.19 0.43((O-carboxymethyl) hydroxylamine

hemihydrochloride2.82 0.05

DL-Vinylglycine 0.47 0.42

DL-Propargylglycine 6.94 0.19

4-Chloromercuribenzoic acid 3.21 0.13Cycloserine 2.27 0.25Penicillamine 5.21 0.54


since it reacts with pyridoxal-linked aminotransferases to form ahighly reactive acylating agent that attacks other active-site resi-dues forming a stable complex [35]. DL-Vinylglycine and DL-propar-gylglycine are also mechanism-based inhibitors of PLP containingenzymes [35–37]. 4-Chloromercuribenzoic acid is a thiol modifyingreagent that can react with free catalytically important cysteineleading to inhibition [38,39]. In some cases, extra PLP can protectthe enzyme from inhibition by this compound [30]. Inhibition bythese six compounds of the aminotransferase activity at 0.45 mMalanine and 0.2 mM 2-oxoglutarate is shown in Table 6. I0.5 valueswere determined for the two most potent inhibitors, O-Carboxy-Methyl hydroxylamine hemihydrochloride and cycloserine, at thesame substrate concentration and found to be 6.9 and 119 lM,respectively. D-Alanine was also tested for inhibition of HvAlaATand none was observed, suggesting that this stereoisomer does notbind to HvAlaAT in a competitive manner (data not shown).

Determination of AlaAT subunit structure by analytical gel filtration

In order to assess the oligomeric state of HvAlaAT, analytical gelfiltration was performed on a Superdex-200 column. A set of pro-tein standards were resolved first, followed by a sample of recom-binant tag-free HvAlaAT under reducing conditions. After fittingto the standard curve generated by the data set from proteins withknown molecular weights, the molecular weight of HvAlaAT wasdetermined to be approximately 106 KDa, in close agreement withthe theoretical size of a protein homodimer (105,761 Da) com-prised of two identical 53 kDa subunits (Fig. 4). This result is consis-tent with other reports that suggest many related aminotransferaseenzymes are homodimers [25,31,32,40].

Table 5Ratio of AspAT/AlaAT activity of various AlaAT forms.

Enzyme Hv AlaAT Os AlaAT Zm AlaAT2

SA (U/mg) AlaAT 3.26 1.43 0.11SA (U/mg) AspAT 0.17 0.07 0.01AspAT/AlaAT 0.10 0.10 0.10

PLP-dependence of barley AlaAT

Although most literature reports suggest that HvAlaAT will uti-lize a pyridoxal 5-phosphate (PLP) cofactor based on homologywith other well-characterized aminotransferase enzymes [40–42], one study suggested that HvAlaAT did not require PLP foractivity based on the lack of activation of enzyme activity uponaddition of PLP to an HvAlaAT assay mixture [31]. However, sincePLP often is covalently bound to the amino group of a Lys residueon the enzyme via a Schiff base linkage, it is common for HvAlaATto be fully loaded with PLP upon purification [40–42]. Because oftheir extended conjugation, PLP Schiff bases absorb in the near-UV region of the spectrum and can be identified spectrophotomet-rically, thus allowing direct examination of the PLP content in aprotein preparation [40–42]. Tag-free HvAlaAT was tested for thepresence of the Schiff base (aldimine) linkage between a conservedLys residue (K299) and a PLP cofactor. Absorbance at 420 nm wasobserved in this enzyme preparation, characteristic of an en-zyme-bound PLP Schiff base (Fig. 5). Treatment of the protein withhydroxylamine to form the PLP-hydroxylamine oxime shifted theabsorbance maxima from 420 to �380 nm as expected (Fig. 5)[41]. This data supports the presence of bound PLP in tag-free HvA-laAT preparations.

Additive/buffer screen and dynamic light scattering forcrystallography

After no suitable crystallization leads were obtained for the na-tive N-terminally-His-tagged HvAlaAT or for any of the variants inTable 2, a buffer and additive screen was performed as described inthe Materials and Methods section. As previously noted, the nativeenzyme, the single site variant K119H, and the penta-substitutedvariant Q30HA,Q33S, Q35A, K37H, and Q39A were selected forthe additive/buffer screen. These two variants were chosen be-cause each initially displayed high polydispersity as assessed byDLS in the original buffer condition of 20 mM Tris buffer pH 8.0,150 mM NaCl, and 2 mM beta mercaptoethanol. DLS data were col-lected after 3 days incubation at 4 �C for each reagent to determine

Zm AlaAT 1 Bh AlaAT Rp D-AlaAT Pp b-AlaAT

1.20 1.14 0.39 0.090.11 1.01 0.33 0.090.10 0.90 0.90 1.00

Fig. 4. Molecular Weight Determination of Tag-free barley AlaAT by Analytical GelFiltration: Standards (diamonds), HvAlaAT (triangle).

Fig. 5. Spectrum of barley AlaAT before (solid line) and after (dotted line) treatmentwith HONH2.

Table 7Data collection and refinement statistics. Values in parentheses are for the highestresolution shell. Rsym = |I� <I>|/I, where I is the observed intensity and <I> is theaverage intensity. Rwork = ||Fo| � |Fc|/|Fo|, calculated using 95% of the data, where |Fo|is the observed structure factor amplitude and |Fc| is the calculated structure factoramplitude. Rfree is the R calculated with 5% of the randomly selected data that wereomitted from the refinement.

Diffraction data statistics

Wavelength (Å) 1.000Space group P21212Unit cell lengths (Å) a = 119.9, b = 127.0, c = 75.6Unit cell angles (�) a = b = c = 90Resolution range (Å) 87.2–2.7(2.8–2.7)Mosaicity (�) 1.35Number of measured reflections 126240Number of unique reflections 32085Redundancy 4.1(4.1)Completeness (%) 97.1(98.8)<I/r (I)> 15.0(2.5)Rsym 0.10(0.49)

Refinement statisticsNumber of reflections

Working set 29484Test set 2601Rwork/Rfree (%) 21.6/27.9

Molecules per asymmetric unit 2Number of atoms

Protein 7410Cycloserine-PLP adduct 44Water oxygen 61

Average B-factor (ÅA0

2)Protein 41.3Cycloserine-PLP adduct 34.3Water oxygen 35.2

Rmsd bonds/angles (ÅA0

/X) 0.024/2.532

Ramachandran plot (%)Most favored 89.8Additionally allowed 8.8Generously allowed 0.5Disallowed 0.8


best buffer conditions. Interesting additives and buffers were se-lected based on the quality of the DLS profile. The primary DLSpeak represented more than or equal to 90% of the scattered lightand the sample polydispersity was below 15%. The best buffer wasfound to be pH 10 carbonate, and useful additives were the co-fac-tor PLP, the inhibitor L-cycloserine, or the reducing agent DTT. Astorage buffer containing all these components resulted in mono-disperse DLS behavior for all three protein samples.

Crystallization, X-ray data collection, structure determination, andrefinement of HvAlaAT-K119H

The HvAlaAT-K119H protein sample in optimal solution condi-tions yielded protein crystals. Optimization of these conditions re-sulted in X-ray quality crystals. X-ray data collection and structurerefinement statistics are listed in Table 7. Data reduction indicatedthat the crystals possessed an orthorhombic, P21212 lattice. Know-ing the lattice extents, the Matthews parameter [43] was calcu-lated to be 5.22 Å3/Da for one molecule in the crystallographicasymmetric unit, which corresponds to 76% solvent, or 2.61 Å3/Da for two molecules in the asymmetric unit, which correspondsto 53% solvent. Since the latter situation is more probable, weassumed that the HvAlaAT-K119H crystals contained two mole-cules in the asymmetric unit prior to the molecular replacement(MR) work.

The MR solution consisted of a dimer in the asymmetric unit. Atthe time of this work, the P. furiosis AlaAT structure, PDB entry

1XI9, was the most identical one available; since then, the more se-quence identical human AlaAT-2 structure, PDB entry 3IHJ, has beendeposited in the PDB. A sequence alignment containing the barley,human and P. furiosis enzymes is displayed in Fig. 6. As can be seenin the alignment, the HvAlaAT-K119H sequence is significantly lar-ger and has numerous insertions relative to the P. furiosis enzyme.The initial MR density map was weak in the gap areas, but the two-fold density averaging with program RESOLVE improved the densityenough to allow unambiguous residue placement.

The final barley AlaAT structure contains residues 3–481 formolecules A and B of the enzyme, 61 water molecules, and L-cyclo-serine-PLP adducts (one per active site). A ribbons drawing of thisdimer is shown in Fig. 7. The first 11 residues of the barley AlaATconstruct contain a deca-histidine tag, and since His-tags are rarelyordered in protein structures, it is not surprising that the immedi-ate N-terminus is disordered in this structure. Covalent cycloser-ine-PLP derivatives have been observed previously; in the crystalstructure of D-amino acid aminotransferase (PDB entry 2DAA), acovalent adduct forms between D-cycloserine and PLP [44].

The final structure has respectable R-factors (Rwork = 21.6%;Rfree = 27.9%) and stereochemistry (Table 7). The Ramachandranplot quality is also quite good for a moderate resolution structuresuch as this; 89.8% of the residues are in the most-favored region,8.8% are in the additionally-allowed region, and 0.5% are in thegenerously-allowed region. Though 0.8% or 8 residues are in thedisallowed region, it is worth noting that these residues haveRamachandran values near allowed values.

Barley AlaAT possesses a ‘‘PLP-dependent transferase’’ fold simi-lar to that of aspartate aminotransferase (AspAT) TM1255 from T.


maritime (PDB entry 1O4S)[45]; a ribbons figure colored to reveal thecharacteristic domain structure is shown in Fig. 8. Barley AlaAT pos-sesses an N-terminal arm (residues 3–223), a small ab-domain (res-idues 24–64 and 356–481) dominated by helices, and a large abadomain (residues 65–355). The active site region which containsthe PLP adduct lies between these two domains. A noteworthy fea-ture of the barley AlaAT structure is the presence of a C51–C453disulfide (Fig. 8), which is not conserved in P. furiosis AlaAT or in hu-man AlaAT2. The disulfide is formed in molecule A of barley AlaATbut not in molecule B and this is likely due to the use of DTT in pre-paring the protein sample for crystallization. A superposition of thehuman AlaAT2 and barley AlaAT structures, as displayed in Fig. 9,highlights how structurally different these two enzymes are in theirN-terminal regions.

The barley AlaAT monomer structure displayed in Fig. 8 showshow the PLP-L-cycloserine adduct is bound in an active site pocketbetween the small and large domains. A network of hydrogen-bonded interactions envelop the PLP-cycloserine adduct in eachactive site, and these are shown in Fig. 10 for molecule A of the bar-ley AlaAT dimer. The 501a (‘‘a’’ for molecule A of the dimer) PLP ad-

Fig. 6. Sequence alignment of HvAla-AT-K119H, human alanine aminotransferase-2 (

duct binds above Tyr174a and below Val260a, and is stabilized byten hydrogen-bonds with protein residues in the active site; nineinteractions involve residues within molecule A and one involvesTyr112b from a neighboring molecule B AlaAT molecule at the di-mer interface. At one end of the 501a adduct, the phosphate moietyalone engages in six hydrogen bond interactions; these includebonds with the amide nitrogen atoms of Ala148a (2.9 Å) andSer149a (3.1 Å), as well as with the side chain nitrogen atom ofArg308a (2.5 Å), and finally with the side chain oxygen atoms ofSer296a (2.7 Å), Ser298a (3.3 Å), and Tyr112b (2.6 Å) of moleculeB in the dimer. Moving clockwise around the PLP-cycloserine ad-duct, the PLP six-member ring nitrogen hydrogen bonds to a sidechain oxygen of Asp258a (2.5 Å), the hydroxyl oxygen off the PLPring bonds to the side chain nitrogen of Asn230a (2.7 Å), and thecarbonyl oxygen of the 5-member L-cycloserine portion of the ad-duct participates in a hydrogen bond with a side chain nitrogen ofArg452a (2.9 Å). The barley AlaAT catalytic lysine, K299a, whichforms a Schiff base with the aldehyde group of the PLP co-factorvia its e-amino group [45] in the transamination reactions of theenzyme, lies above the adduct in Fig. 10, and in this structure,

PDB entry 3IHJ), and alanine aminotransferase from P. furiosus (PDB entry 1XI9).

Fig. 7. Ribbon diagram of the barley AlaAT dimer with the 2-fold symmetry axisperpendicular to the plane of the figure. Molecule A is green and molecule B is blue.The PLP-L-cycloserine adducts in each active site and the His119 side chains in eachmonomer, which were mutated from Lys in the constructs which crystallized, arealso displayed in sphere mode; for these, the carbon atoms are rendered in yellow formolecule A and in pink for molecule B, with all other atoms using default colors. TheC51–C453 disulfide, with S atoms colored yellow, is also rendered in sphere mode.

Fig. 9. Ribbon diagram of a superposition of human AlaAT 2 (pink with a magentaN-terminus; PDB entry 3IHJ) with the barley AlaAT monomer (cyan with dark blueN-terminus). The fold of the two structures is quite similar except for the N-termini.

Fig. 8. Ribbon diagram of barley AlaAT molecule A colored to highlight the differentdomains in the structure: N-terminal arm (dark blue), small domain (red), largedomain (cyan) The PLP-L-cycloserine adduct in the active site region and C51-C453disulfide are also displayed.

Fig. 10. The binding interactions of the PLP-L-cycloserine adduct (501a) inmolecule A of the barley AlaAT structure. Hydrogen-bonded interactions arerendered as dashed lines. The carbon atoms of molecule A are rendered as green andthe carbon atoms of molecule B are rendered as blue.


the e-amino nitrogen makes a polar contact of 3.2 Å with nitrogenatom that bridges L-cycloserine with PLP in the adduct. A numberof aromatic and hydrophobic residues define the binding pocket.Residues which define the roof of the binding pocket along withVal260a, include Tyr261a, Ser296a, Ser298a and Tyr398a. Residueswhich define the floor or base of the binding pocket in Fig. 11 alongwith Tyr174a include Tyr19a, Val21a, Leu176a, and Tyr177a. Ile226a defines the backside of the pocket, and Ile53a, Gly54a,Cys305a and Leu333b, along with Tyr112b help define the frontborder of this binding pocket.

Discussion

Recent evidence provides a connection between improved plantgrowth and enzymes involved indirectly in nitrogen uptake orutilization, including glutamine synthetase (GS), glutamate syn-thase (GOGAT), asparagine synthetase (AS), glutamate dehydroge-nase (GDH), and aspartate aminotransferase (AspAT) [46–52].Collectively, these results suggest an important role for nitrogen up-take and utilization in determining yield. Transgenic expression ofbarley AlaAT increases yield in rice and canola [6,7]. Given that Ala-

Fig. 11. The molecule A active sites of barley AlaAT (yellow carbon atoms) and P.furiosis AlaAT (PDB entry 1XI9; blue carbon atoms) superimposed. Barley AlaATcontains the PLP-cycloserine adduct in the active site while the PfAlaAT simplycontains PLP.


AT effectively enhances yield under a variety of soil nitrogen levels,it seems likely that HvAlaAT is enhancing the nitrogen use efficiency(NUE) of the plant, similarly to the other NUE leads described above.

In order to elucidate the mechanism of HvAlaAT action severalfactors must be considered. In particular it is necessary to knowthe reaction direction, expression pattern, and biochemical charac-teristics of the enzyme. Since the reaction catalyzed by AlaAT ishighly reversible, it is plausible that in vivo the reaction occurs ineither direction. We have carefully characterized the kinetics in or-der to determine if in vitro analysis would suggest a preferred reac-tion direction. While the reaction seems to slightly favor alaninein vitro, the direction of the reaction in vivo will be determinedby the concentrations of the various reaction substrates in themicroenvironment of the enzyme due to the calculated equilib-rium constant (Keq) of the reaction near 0.5. Further analysis ofreaction flux using identifiable isotopes is necessary to clearly de-fine the reaction direction in various tissues in wild-type andtransgenic plants.

Here we report the first detailed kinetic and physical character-ization of a purified plant alanine aminotransferase, along with acomparison between barley alanine aminotransferase and alanineaminotransferase from several other plant and bacterial species.The values of kcat and Km vary for the different AlaAT enzymesand no obvious, consistent kinetic differences for the primary sub-strates of the reaction (pyruvate, 2-oxoglutarate, glutamate, andalanine) were observed.

The equilibrium constant for the reaction catalyzed by barleyAlaAT was calculated from our kinetic values using the Haldaneequation [53] to be approximately 0.51 in the direction of glutamateformation. This compares well to various determined Keq values forthis reaction between 0.5 and 2.0 from the NIST standard referencedatabase 74 (for thermodynamics of enzyme catalyzed reactions).The Keq for HvAlaAT in the glutamate forming direction also sug-gests that this enzyme shows a small preference for Ala formation.

This report also provides the first detailed determination of thesubstrate specificity of a plant alanine aminotransferase. Thebarley enzyme primarily interconverts glutamate and alanine.While other interconversions can be observed in vitro, they aresubstantially less efficient although the in vivo situation may bequite different. AspAT activity was the second most efficient reac-tion for the barley enzyme and this is consistent with other alanineaminotransferase enzymes [25]. When we tested the other AlaATenzymes for this activity they fell into two classes: (1) the plant en-

zymes with very low AspAT activity and (2) the bacterial ones thathave considerable AspAT activity. Considering that these enzymesare annotated as D or b-alanine aminotransferase it is likely theycan utilize a number of substrates and should, in fact, be consid-ered non-specific aminotransferases.

We have shown that, similar to other reported aminotransfer-ases, barley alanine aminotransferase uses PLP as a cofactor andhas a bi–bi ping–pong reaction mechanism. This result is especiallyimportant because of an earlier report that suggested that HvAlaATdid not use PLP [31]. The evidence that barley AlaAT uses PLP isthreefold: (1) AlaAT has the spectral properties associated with en-zyme bound to PLP (Fig. 5), (2) various PLP antagonists and amino-transferase inhibitors act against barley AlaAT (Table 6), and (3) thecrystal structure clearly reveals PLP intimately bound to the en-zyme in the form a covalent adduct with L-cycloserine. Since the ef-fect of adding PLP to purified AlaAT varies, it is likely that in mostcases the extracted enzyme is not fully loaded with PLP. In additionsubstrate titrations with and without PLP confirm that PLP loadingaffects only the kcat and not the Km of the enzyme, consistent with arole for PLP in the chemistry of the reaction performed by AlaAT(data not shown). Finally, as reported for all other alanine amino-transferases, the barley enzyme is a dimer with a subunit molecu-lar weight of �53 KDa. The crystal structure also reveals anassociated dimer and a PLP binding pocket with stabilizing interac-tions involving protein residues from both monomers.

We have studied the characteristics of a number of AlaAT formsin order to determine if the physical or kinetic properties of the en-zyme could be improved and if so whether or not the improvementin the enzyme would lead to a further increase in the yield pheno-type. Although no major differences in kinetic competence wereobserved in the panel of alanine aminotransferases tested here,some subtle variations could potentially impact this enzyme’s per-formance in transgenic crops. In particular, several of the bacterialenzymes had lower Km values, especially for pyruvate which mightbe advantageous under conditions where pyruvate is limiting.However, none of the other enzymes tested had substantially high-er kcat than HvAlaAT. Indeed, since other AlaAT orthologs have beencharacterized with much higher Vmax [25] or lower Km values [27](Table 3); clearly there is room to engineer a kinetically superiorversion if necessary.

Since pyruvate levels are very low and possibly limiting in plan-ta, increasing the binding strength for this amino acceptor mightincrease flux through this reaction. Additionally, since there is noevidence that the reaction is at equilibrium even in transgenicplants, improving the Vmax of the enzyme might increase flux andtherefore NUE. However, it is worth noting that since the substratespecificity of barley AlaAT is much tighter than that of the bacterialenzymes, maintaining this tight specificity for alanine might benecessary. Examination of the physiological characteristics oftransgenic plants containing a variety of these genes will bestdetermine the most important characteristics of the enzyme forimproving crop NUE and yield.

Solving the barley AlaAT enzyme X-ray crystal structure vali-dated our strong initial convictions and our biochemical finding thatthe enzyme is a dimer and requires PLP. The structure represents thefirst plant AlaAT enzyme to be solved by crystallography, and it con-firms that barley AlaAT possesses a PLP-dependent transferase foldobserved in other AspAT and AlaAT enzyme crystal structures.

The barley Ala-AT crystal structure contains a PLP-L-cycloserineadduct in the active site, and therefore it is not surprising that thebest stabilizing buffer for crystallizing the enzyme would containPLP and L-cycloserine. While it is less obvious why only theK119H variant of barley AlaAT would yield crystals suitable forstructural studies, the K119H-barley AlaAT structure does provideanother example of how rational surface engineering [13,54] canbenefit protein crystallography. Examination of the structure


clarifies that residue 119 is indeed on the surface of the enzyme(Fig. 7); this was something we did not know prior to solving thestructure. Careful crystal packing analyses reveal that the His119side chain is not engaged in any noteworthy intermolecularcontacts in this structure, but is solvent-exposed and lies on a helix(Fig. 7). Replacing the more floppy Lys side chain with a less floppyHis side chain should lead to surface entropy reduction in thislocation, which was the intent of this proposed mutation, and suchmutations have been known to benefit crystallization [13]. It is alsonot unreasonable to believe that good fortune may have factoredinto the crystallization success of the K119H variant relative toall the other tested constructs.

As has been noted in the paper, barley AlaAT is much more spe-cific for utilizing alanine as a substrate than other AlaAT enzymes,such as the bacterial P furiosus AlaAT, which displays significantactivity with alanine, glutamate, and aspartate as substrates [25].With crystal structures now available for both these enzymes, itis worthwhile to examine a superposition of the active site regionof P. furiosis AlaAT (PfAlaAT, PDB entry 1XI9) onto the active site re-gion of barley AlaAT to search for insights regarding this substrateselectivity; this superposition is presented in Fig. 11. L-cycloserineis a structural analog for L-alanine, so the binding region around L-cycloserine in the PLP-cycloserine adduct of the barley structurecan shed some insights on the binding region for the Ala substrate.An examination of Fig. 11 reveals that while key active site pocketresidues between the barley and PfAlaAT enzymes are very similarin nature and spatial location in the binding cavity for PLP, on theleft-half of the active site region, in the binding region aroundcycloserine, on the right-half of the active site, the key active sitepocket residues in the barley enzyme come much closer to cyclo-serine than in PfAlaAT. The Ile53 side chain in the barley enzymeis significantly closer to the cycloserine moiety than that of thehomologous Ile37 of PfAlaAT; the CG1 atom of HvAlaAT Ile53 is3.2 Å from the OG atom of cycloserine, while the nearest side chainatom in PfAlaAT Ile37 is 5.7 Å away. Moreover, in the barleyenzyme, the Val21 and Arg22 side chains help define the lowerright binding cavity and lie on a helix; the corresponding stretchof peptide in PfAlaAT, residues 14–20, is disordered, and thuspossibly flexible and ill-defined. Finally, two other key residuesthat define Ala binding pocket in the barley enzyme, Arg452 andTyr19 come slightly closer to cycloserine than the correspondingresidues in PfAlaAT, Arg371 and Tyr13. All in all, the binding regionaround L-cycloserine in barley AlaAT, where an Ala substratewould likely bind, would appear to favor the binding of smallside chain substrates like such as Ala over larger side chain aminoacids like Asp because key side chains that border this region, mostnotably Ile53 and Val21, create steric constraints for the binding oflarger amino acid substrates. The PfAlaAT enzyme, on the otherhand, has a larger, more open and less sterically-constrainedbinding cavity in the region where amino acids would bind, andthis is likely why PfAlaAT is active with glutamate, aspartate, andalanine.

Acknowledgments

X-ray data were collected at the Southeast Regional Collabora-tive Access Team (SER-CAT) _22-ID beam line at the AdvancedPhoton Source, Argonne National Laboratory. Use of the AdvancedPhoton Source was supported by the US Department of Energy,Office of Science, Office of Basic Energy Sciences, under ContractNo.W-31-109-Eng-38. The authors would like to thank XiaoyunWu for isolating the barley AlaAT gene from seedlings.

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the enzymology of alanine aminotransferase (alaat) isoforms from hordeum vulgare and other...

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