Biochemical and structural characterization ofCYP124: A methyl-branched lipid �-hydroxylasefrom Mycobacterium tuberculosisJonathan B. Johnston, Petrea M. Kells, Larissa M. Podust, and Paul R. Ortiz de Montellano1

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517

Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved September 25, 2009 (received for review July 6, 2009)

Mycobacterium tuberculosis (Mtb) produces a variety of methyl-branched lipids that serve important functions, including modu-lating the immune response during pathogenesis and contributingto a robust cell wall that is impermeable to many chemical agents.Here, we report characterization of Mtb CYP124 (Rv2266) thatincludes demonstration of preferential oxidation of methyl-branched lipids. Spectrophotometric titrations and analysis ofreaction products indicate that CYP124 tightly binds and hydroxy-lates these substrates at the chemically disfavored �-position. Wealso report X-ray crystal structures of the ligand-free and phytanicacid-bound protein at a resolution of 1.5 Å and 2.1 Å, respectively,which provide structural insights into a cytochrome P450 withpredominant �-hydroxylase activity. The structures of ligand-freeand substrate-bound CYP124 reveal several differences induced bysubstrate binding, including reorganization of the I helix andclosure of the active site by elements of the F, G, and D helices thatbind the substrate and exclude solvent from the hydrophobicactive site cavity. The observed regiospecific catalytic activitysuggests roles of CYP124 in the physiological oxidation of relevantMtb methyl-branched lipids. The enzymatic specificity and struc-tures reported here provide a scaffold for the design and testing ofspecific inhibitors of CYP124.

cytochrome P450 � phytanic acid � �-hydroxylation � X-ray structure

Mycobacterium tuberculosis (Mtb) is the causative agent ofhuman tubercular infection that, according to the World

Health Organization (1), results in more than two million deathseach year. Approximately one-third of the world’s populationharbors the bacterium in a latent, noninfective state, and newinfections are occurring at an alarming rate. The emergence ofdrug-resistant and multidrug-resistant Mtb strains has made thefrontline antituberculosis drugs (isoniazid, streptomycin, rifam-picin, ethambutol, and pyrazinamide) less effective. Yet, no neweffective antitubercular drugs have been approved since the1960s, and there is an urgent need to identify new drug targetsto help fight the spread of Mtb and quell the rising mortality ratesassociated with infection.

Mtb produces a rich array of lipids that, among other things,allow it to thrive in the harsh environment of the macrophage,confer resistance to a variety of chemical agents, and stimulatethe host immune response during pathogenesis. A large portionof the Mtb genome encodes genes involved in lipid biosynthesisand metabolism, including 20 putative cytochrome P450 en-zymes that are of interest as potential drug targets (2–5). Five ofthe 20 Mtb P450 enzymes (CYP51, CYP121, CYP125, CYP130,and CYP142) have been reported in purified form (2, 3, 6–14).To date, only CYP51 and CYP121 demonstrate a definedcatalytic activity (14, 15). More than 10 years elapsed betweenthe sequencing of the Mtb genome and the association of acatalytic activity with a second orphan P450 enzyme—CYP121(15). Importantly, the recent breakthrough with CYP121 camein part from knowledge of the function of its f lanking gene (15,16). Catalytic functions are difficult to assign to the remainingMtb P450s because they have diverged significantly from P450

enzymes of known function, and their organization within theMtb genome provides few clues about their potential biologicalroles (2, 3, 17, 18).

CYP124 is found in pathogenic and nonpathogenic mycobac-teria species, actinomycetes, and some proteobacteria, whichsuggests that it has an important catalytic activity (2). CYP124(Rv2266) is located adjacent to a three-gene operon containinga sulfotransferase (Sft3, Rv2267c) that catalyzes the PAPS-dependent sulfation at the �-position of menaquinone MK-9DH-2 (19, 20). CYP128 (Rv2268c) is thought to hydroxylate the�-position before sulfation (19). The sulfated form of the lipid,termed ‘‘S881,’’ is associated with the outer cell membrane ofMtb, where it acts as a negative modulator of virulence in themouse model of infection (20). We postulated that CYP124might have a related substrate, i.e., a lipid with repeating methylbranching due to its proximity to the Sft3 operon. We describehere the biochemical characterization of CYP124 that includesidentifying a series of substrates consistent with �-hydroxylaseactivity and, importantly, a marked preference for lipids con-taining methyl branching. We also report high-resolution struc-tures of the ligand-free and phytanic acid-bound forms ofCYP124, the first structures of a native cytochrome P450 thatprimarily oxidizes the chemically disfavored �-position of ahydrocarbon chain.

ResultsSpectroscopic Characterization of CYP124. Purified CYP124 (Fig. S1in SI Appendix) is in the ferric low-spin six-coordinated form, asjudged by the UV-visible absorption spectrum that shows a largepeak at 421 nm and smaller peaks at 538 and 571 nm corre-sponding to the �- and �-bands, respectively (Fig. 1). FerricCYP124 underwent facile reduction to the ferrous form bytreatment with sodium dithionite, which generated a spectrumwith peaks at 415 and 544 nm. Ferrous CYP124 in complex withCO, however, showed the signature Soret band at 450 nm as wellas a smaller peak at 555 nm. Incubating ferric CYP124 withvarious methyl-branched lipid substrates (see below) shifted theheme to the high-spin form, as indicated by the appearance of adominant peak at 395 nm, small peaks at 511 and 544 nm, anda charge-transfer band at 646 nm (Fig. 1).

CYP124, like most P450 enzymes (21), binds azole drugsthrough coordination to the heme iron to produce characteristicType-II spectra with a peak between 425 and 435 nm and a broad

Author contributions: J.B.J., L.M.P. and P.R.O.d.M. designed research; J.B.J., P.M.K., andL.M.P. performed research; J.B.J., L.M.P., and P.R.O.d.M. analyzed data; and J.B.J., P.M.K.,L.M.P., and P.R.O.d.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 2WM4 and 2WM5).

1To whom correspondence should be addressed: E-mail: ortiz@cgl.ucsf.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907398106/DCSupplemental.

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trough at 390–410 nm that reflect azole coordination to theheme iron through a nitrogen atom (22). Dissociation constantsfor CYP124 with azoles (clotrimazole, KD � 2.5 � 0.1 �M;econazole, KD � 2.1 � 0.1 �M; miconazole, KD � 1.9 � 0.2 �M)were obtained from the concentration-dependent production ofType-II difference spectra (Fig. S2 in SI Appendix). On the otherhand, cytochrome P450 enzymes typically bind to substrates byejecting the axial heme water ligand, which gives rise to Type-Ibinding spectra with a peak at 385–390 nm and a trough centeredat 420 nm (22). Bifonazole bound as a Type-I ligand anddisplayed the highest affinity toward CYP124 (KD � 494 � 48nM). The heme spin-state of CYP124 was not detectably per-turbed when titrated with fluconazole.

We titrated CYP124 with a variety of lipids including isopre-noids, linear and branched fatty acids, and branched alkanes(Table 1). Several of these lipids formed tight complexes withCYP124 as indicated by the resulting concentration-dependentType-I difference spectra (Table 1 and Fig. S3 in SI Appendix).The binding affinities of CYP124 toward lauric (�100 �M),palmitic (�100 �M), 15-methyl palmitic (1.01 �M), and phytanic(0.22 �M) acid increase with methyl branching. On the otherhand, the decrease in affinity toward pristane (205 �M) andphytane (178 �M) supports the argument that a polar functionalgroup is necessary for key interactions within the active site.Moreover, the relative affinities of geraniol, farnesol, geranylge-raniol, and farnesyl diphosphate show that longer methyl-branched lipids bind tighter to the enzyme and that the type offunctional group is important for binding, whereas the degree of

unsaturation does not contribute significantly to binding. Theproximity of the CYP124 gene to the Sft3 operon led us to alsotest phylloquinone and menaquinone as ligands of CYP124, butwe were unable to detect binding.

CYP124 Catalyzes �-Hydroxylation of Methyl-Branched Lipids. Basedon the Type-I spin shifts and high-affinity binding towardmethyl-branched lipids, CYP124 was incubated with spinachferredoxin, spinach ferredoxin-NADP�-reductase, various lip-ids, and NADPH, and the reaction products were compared byGC-MS with those obtained in control reactions in which eitherCYP124 or NADPH was omitted. New signals appeared in theGC chromatograms that depended on the presence of bothNADPH and CYP124 in the reaction mixture (Fig. 1 and Fig. S4in SI Appendix). In each case, the trimethylsilylated (TMS)metabolites eluted from the GC at a higher temperature than therespective substrates, which is consistent with the presence of aTMS-protected alcohol in each metabolite. The reaction prod-ucts, identified by mass spectrometry using characteristic mo-lecular ion and fragmentation patterns, confirm that CYP124oxidation occurs primarily at the �-position (Fig. S4 in SIAppendix). CYP124 converted phytanic (Fig. 1) and 15-methylpalmitic acid (Fig. S4 in SI Appendix) each into a single product.and their molecular ions at m/z � 472 and 430, respectively,confirm the presence of an additional TMS-protected alcohol ineach. The fragment ion at m/z � 103 corresponds to the loss of-CH2OSi(CH3)3 from the �-position of a saturated branched-lipid with a TMS-protected hydroxyl group (23), and we ob-served such fragments with the new phytanic and 15-methylpalmitic acid metabolites (Fig. S4 in SI Appendix).

Isoprenoids (farnesol, farnesyl diphosphate, geranylgeraniol)were also efficiently oxidized by CYP124 into the respective�-hydroxylated products (Fig. S4 in SI Appendix). These assign-ments were based on comparisons with the product formed inincubations of farnesol with CYP2E1, which catalyzes �-hydroxylation of farnesol (24). Both the retention times and massspectra of the CYP124 and CYP2E1 metabolites matched in ourassays. The product from farnesyl diphosphate was treated withalkaline phosphatase before GC-MS analysis to release thefarnesol structure, which then matched the mass and retentionpattern seen for the metabolite of farnesol itself. The geranylge-raniol metabolites show the mass increase and fragmentationpattern characteristic of terminal oxidation adjacent to an allyliccarbon (Fig. S4 in SI Appendix). Incubation of CYP124 withgeranylgeraniol also produced geranylgeranic acid and �-hydroxylated geranylgeranic acid (Fig. S4 in SI Appendix),indicating that two catalytically competent binding modes arepossible with this substrate in the active site of CYP124.

Palmitic acid binds with a sharply decreased affinity and isoxidized �200-fold less effectively than analogous methyl-branched lipids (Table 1). Moreover, CYP124 oxidized the �-1,�-2, and �-position of palmitic acid (Fig. S4 in SI Appendix),whereas we did not observe �-1 metabolites with any of themethyl-branched lipids (Fig. S4 in SI Appendix), nor were anyoxidized metabolites detected from incubating CYP124 withgeraniol, lauric acid, phytane, pristane, phylloquinone (vitaminK1), menaquinone (vitamin K2), or bifonazole. The majorreaction we observed with CYP124 is �-oxidation of methyl-branched lipids.

Overall Structure of CYP124. The structures of the ligand-free (1.5Å) and substrate-bound (2.1 Å) CYP124 are similar with anRMSD value for C� carbons of �0.9 Å when superimposed. Thestructural features commonly involved in heme binding, sub-strate recognition, and formation of the active site cavity arereadily distinguished (Fig. 2), and are comparable with otherP450 enzymes for which structures are known (7, 11, 13, 25). The‘‘f loor’’ of the active-site cavity is formed by heme that is

Fig. 1. Biochemical characterization of CYP124. (A) UV-visible absorbancespectra of CYP124 in the ferric (black), ferrous (green), ferric high-spin (blue)and ferrous-CO (red) forms. (B) Overlaid GC chromatograms showing the totalion current (TIC) versus retention time for reactions containing Fdx, Fdr,NADPH and phytanic acid incubated for 10 min in the presence (dotted line)or absence (solid line) of CYP124. The reaction scheme depicts the CYP124- andNADPH-dependent conversion of phytanic acid into �-hydroxy-phytanic acid.Reactions were extracted, and these compounds were derivatized with BSTFAbefore GC analysis, yielding peaks at 18.2 min (S) and 20.7 min (P) correspond-ing to the trimethylsilyl-ester of phytanic acid and TMS-�-hydroxy phytanicTMS-ester, respectively.

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positioned between the I and L helices, with the proximalCys-379 thiolate anchoring the cofactor to the L helix. One halfof the active site cavity is composed of the F, G, and C helices

as well as residues from the BC and FG loop regions. The otherhalf of the active site cavity is roughly defined by the C-terminalloop (Leu-413–His-418) that trails the L helix, as well as fourantiparallel strands comprising �-sheet 1.

Substrate-Induced Conformational Changes. There are importantdifferences between the ligand-free and phytanic acid-boundCYP124 structures. Substrate binding induces a reorganizationof secondary structure elements, as shown in Fig. 2, which arelargely confined to the regions typically involved in forming theactive site and substrate recognition region (25). The BC-loop(Thr-100–Phe-107) moves �5.3 Å toward the G helix, whichpositions Leu-103 and Phe-107 to interact with the C-18 methylbranch of phytanic acid. The D and C helices move in concerttoward the G helix, with N-terminal portions of the D helix(Lys-132–Ala 139) bending toward the active site and theproximal side of the heme. The G helix (Phe-209–Val-231) shifts4.9 Å toward a newly ordered turn between the first and secondstrands of �-sheet 1, positioned across the active-site cavity thatforms a lid over the active site. Importantly, Phe-209 andPhe-212 face into the active site along the hydrophobic backboneof phytanic acid. Residues of the FG loop (Gly-199–Asp-208)and F helix (Lys-184–Gly-199) are positioned toward the activesite in the phytanic acid-bound structure. Substrate binding alsocauses the EF loop (Met-180–Lys-184) to move inward, makingthe structure more closed by �2.9–3.1 Å. Three additionalregions, the GI loop, H helix, and HI loop, all relocate in unisonupon substrate binding, and these movements accompany therelocation of the G helix as it closes the active site. In theligand-free CYP124 structure, �-helical structure is missing fromthe middle of the I helix, whereas in the substrate-bound form,the I helix is continuous.

Table 1. CYP124 binds and hydroxylates methyl branchedlipids

Compound Chemical structure Dissociation constant

KD, µM Specific activity*

Michaelis constant KM, µM

Lauric acid HO

O

>100† n.d. n.d.

Palmitic acid HO

O

>100† 0.07 ± 0.03‡ n.a.

15-Methyl palmitic acid HO

O

1.01 ± 0.07 7.6 ± 1.5 9 ± 4

Phytanic acid HO

O

0.22 ± 0.006 9.9 ± 2.7 54 ± 8

Arachidic acid HO

O

n.d. n.d. n.d.

Phytane

205 ± 14 n.d. n.d.

Pristane

178 ± 18 n.d. n.d.

Geraniol HO

25 ± 1.8 n.d. n.d.

Farnesol HO

1.04 ± 0.05 15.5 ± 2.8 36 ± 3

Geranylgeraniol HO

0.48 ± 0.06 9.6 ± 3.1 32 ± 4

Farnesyl diphosphate OPOPHO

OO

OHOH 90 ± 13 4.8 ± 0.9 n.a.

n.d., not detected; n.a., not available, poor solubility prevented reaching saturation.*Units of (nmol of product � min�1 � nmol of CYP124�1).†Value indicates an estimated lower limit.‡The reported error values are standard deviations.

Fig. 2. Superimposed structures of ligand-free and phytanic acid-boundCYP124 are shown with the common P450 secondary structure elementslabeled. The protein backbone is depicted by colored ribbon and the heme byblack sticks. Amino acid side chains and phytanic acid are omitted for clarity.Shown in light blue are those regions of CYP124 that do not significantlychange upon substrate binding. The yellow (ligand-free) and red (phytanicacid-bound) structure elements undergo conformational change upon sub-strate binding.

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Structure of the CYP124 Active Site with Phytanic Acid Bound. Theactive site of ligand-free CYP124 shows the strictly conservedCys-379 coordinated to the heme iron (2.3 Å), water128 (2.2 Å)as the distal ligand, and the iron atom in the plane of theporphyrin ring. Many water molecules are ordered in the ligand-free active site consistent with the more open conformationfavored in the absence of substrate. Cys-379 also serves as theproximal ligand (2.4 Å) with phytanic acid bound in the activesite, but the iron atom moves out of the plane of the porphyrinring toward the proximal thiolate. Importantly the terminalmethyl group of phytanic acid is 3.8 Å away from the high-spinheme iron and poised for �-oxidation (Fig. 3).

Residues of the active site provide hydrophobic and polarinteractions to bind the methyl-branched lipid chain and car-boxylic acid group, respectively. Fig. 3 shows an expanded viewof phytanic acid bound in the active site of CYP124 andhighlights key residues within 5 Å of the substrate. The reorga-nization of the BC loop and C helix provides many hydrophobicinteractions to the substrate from Ile-94, Leu-103, Phe-107, andIle-111 as well as polar interactions with the hydroxyl of Thr-95and carboxamide of Asn-97. The newly ordered �-sheet 1 turnforms a ‘‘lid’’ over the active site and also binds the substratethrough Ile-58, Leu-60, and Phe-63. The reorganization of theFG loop region positions several hydrophobic residues (Ile-197,Leu-198, and Phe-200) to bind the hydrophobic lipid. Themovement of the G helix shifts Phe-212 to bind phytanic acid. Sixother residues are in the 5- to 6-Å sphere of the substrate andstabilize substrate binding, including Val-315, Tyr-317, Met-318,Phe-416, and Ile-417. Hydrophobic residues of the I helix,including Ile-262, Leu-263, Val-266, and Ala-267, also bindphytanic acid within the active site.

The volume of the active-site cavity with phytanic acid re-moved is 1,370 Å3. Two pockets of this cavity, 300 Å3 and 200 Å3,are not occupied by phytanic acid (Fig. 4), suggesting additionalspace available for binding the true substrate. Hydrophobicresidues Leu-29, Trp-32, Ile-58, Leu-60, Leu-198, Ile-413, Ile-417, and Phe-200 and polar residues Ser-414 and Gln-415constitute the borders of the larger 300 Å3 pocket toward thecarboxylic end of the phytanic acid molecule.

DiscussionAssigning in vivo functions or demonstrating in vitro catalyticactivity with the P450 enzymes of Mtb is not trivial (2, 3, 5, 14,15). The location of CYP124 within the genome of Mtb next toan operon coding for an important sulfotransferase (19) led usto initially explore methyl-branched lipids as substrates ofCYP124 to determine whether it might have a related function.The tight binding affinity and �-hydroxylase activity of CYP124toward a series of lipids indicate that the enzyme preferentially

metabolizes methyl-branched lipids and oxidizes the chemicallydisfavored �-position. Cyp124 is among a group of differentiallyexpressed Mtb genes during infection in the mouse lung (26), andthe CYP124 gene is also conserved in many actinomycetes andproteobacteria, which suggests that the enzyme catalyzes animportant reaction. Work is ongoing to more precisely addressthe in vivo function of CYP124 that includes using gene knock-outs and lipidomics.

Regardless of whether CYP124 is involved in biosynthesis ofthe S881 sulfolipid, it clearly has an activity toward methyl-branched lipids, and Mtb is replete with such lipids that areinvolved in a variety of important and cryptic functions. The Mtbisoprenoid biosynthetic pathway is essential (27) and generatesimportant respiratory menaquinones (28), sulfated forms ofwhich negatively regulate the immune response in mice infectedwith Mtb (19, 20). In fact, CYP124 oxidizes farnesyl diphosphate(FPP), a precursor of longer-chain isoprenoids that are found inMtb (29, 30); however, at this point, we cannot link CYP124 within vivo activity toward FPP. Decaprenyl phosphates are essentiallipid and sugar carriers in the assembly of the mycolic acid–peptidoglycan–arabinogalactan (mAGP) complex of the cell wall(31). Mtb also presents several lipids on its cell surface that havemethyl branching, including sulfolipid-1 (SL-1), di- and polyacyl-treheloses (DAT and PAT), mannosyl-�-1-phosphomycoketide(MPM), phthiocerol dimycocerosate (PDIM), and mycolic acids(32–34). In addition, Mtb, like other actinomycetes, is capable of

Fig. 3. Expanded stereoscopic view of CYP124 with phytanic acid (yellow) bound in the active site. Residues within 5 Å from phytanic acid (green) are shown.The protein backbone is omitted for clarity, and the heme cofactor is shown in orange. Phytanic acid was modeled into the electron density depicted as bluewire mesh.

Fig. 4. Active site of CYP124 in which the surface of the cavity is shown ingray, heme is in orange with the Fe atom in magenta, and phytanic acid ishighlighted in green with the carboxyl group in red. Unoccupied pockets arelabeled by their volumes.

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deriving energy from carbon sources with methyl branching (35,36). In mammals, �-oxidation of branched lipids, such as phy-tanic acid, shifts the register of the �-substituted carbon chain sothat �-oxidation can occur (23). CYP124 could conceivablyfunction in oxidative degradation or balancing of Mtb lipid andcarbon pools.

P450 oxidation of hydrocarbons at the �-position is moredifficult than at the internal positions. Experimental and theo-retical studies of the P450 reaction cycle show that H-atomabstraction is the highest energy barrier in the pathway, anddifferences in chemical reactivity between positions are gener-ally a function of the relative strengths of the C–H bonds thatundergo homolytic cleavage, and thus of the relative stabilitiesof the radical intermediates resulting from homolysis (37–40).The catalytic core of P450 enzymes is highly conserved (41), andthe oxidizing species (Compound I, [FeIV � O]��) should also beequivalent for P450 enzymes with different regiospecificities.The parameters that govern the hydroxylation of thermodynam-ically disfavored sites have been explored (42–54). The modelresulting from these studies suggests that P450 �-hydroxylasesrestrict substrate conformation within the active site to positionthe terminal methyl group near the heme while preventing morereactive positions on the substrate from encountering the heme.

The structures presented here are in accord with this model,and provide important insight into how CYP124 catalyzes �-hydroxylation. The terminal methyl group of phytanic acid ispositioned 3.8 Å away from the heme, consistent with the�-regiospecificity we observed. The active site with phytanic acidbound in it is both hydrophobic and conformationally restrictivenear the heme, consistent with a single phytanic acid metabolite.Other P450 �-hydroxylases typically also have a patch of polaramino acid side-chain residues that bind the substrate ‘‘tail’’ (55).CYP124 substrates also require anchoring in the active site by apolar functional group (hydroxy or carboxylic acid) and eitherremoving it or replacing it by a large diphosphate moiety resultsin a large decrease in binding affinity. The CYP124 structurereveals that the carboxylate group of phytanic acid is bound inthe active site near a patch of amino acid side chains with polarfunctional groups; however, the spacing suggests that the nativesubstrate has a slightly longer lipid chain. This polar region,although important for initial binding, does not define theregiospecificity of CYP124. Isoprenoids (farnesol, farnesyldiphosphate, and geranylgeraniol) differing in chain length,binding affinity, and specific activity were all converted into�-hydroxylated products. Geranylgeraniol was oxidized at the�-position and further oxidized to �-hydroxy geranylgeranic acidas well as directly to geranylgeranic acid (Fig. S4 in SI Appendix).

The CYP124 crystal structure with phytanic acid boundsuggests that regiospecific hydroxylation is the result of manyweak interactions that tightly bind and position the substratewithin the geometry of the active site. Indeed, the side chains ofVal-315 and Ile-417 are more than 4 Å away from the terminalbranch, creating enough space for ambiguous positioning of theterminal carbon of the nonbranched lipids. Lack of methylbranching in palmitic acid lends flexibility such that it can sampleconformations amenable for �-1 and �-2 oxidation. The chem-ically more preferable positions become oxidized, but �-oxidation is still observed. Introducing a single methyl branch, asin 15-methyl palmitic acid, results in �100-fold higher specificactivity and at least the same increase in binding affinity. Mostimportantly, �-1 hydroxylation is now excluded in favor of the�-hydroxylation. Binding of one of the two methyls of thebranched hydrocarbon terminus in a specific active-site cavityapparently locks the other methyl in the position required for�-hydroxylation. It is likely that in unbranched hydrocarbons, theterminal methyl is bound in the same cavity, making substrateoxidation inefficient and, when it occurs, nonregiospecific.

The results presented here are of a cytochrome P450 of theCYP124 family, and, importantly, this ortholog is one of 20 P450enzymes that are potential drug targets in Mtb. Our demonstra-tion that CYP124 has �-hydroxylase activity toward methyl-branched lipids provides important clues about its in vivofunction and suggests physiologically relevant substrates for theenzyme. The substrate specificity defined here, together with thehigh-resolution structures, provides a scaffold for design andtesting of specific CYP124 inhibitors.

Materials and MethodsCYP124 Cloning, Overexpression, and Purification. CYP124 (Rv2266) was am-plified from Mtb H37Rv genomic DNA, as described in ref. 7, by using asense-oligo (TTT TTT CAT ATG CAT CAC CAT CAC CAT CAC GGC CTC AAC ACGGCG ATC) to introduce a unique NdeI restriction site and a six-histidine affinitytag. The antisense DNA oligo (AAA AAA AAG CTT ATT ATT AGG ACC ACG TAACTG GCA GCG TC) added a unique HindIII restriction site. The CYP124 gene wasverified and then ligated into the pCW expression vector (6, 7), and theenzyme was overexpressed in Escherichia coli DH5� as described in ref. 7.CYP124 was purified by using Ni-NTA2� followed by ion-exchange chroma-tography steps as described in ref. 7.

Spectrophotometry. UV-visible spectra were recorded on a Cary dual beamspectrophotometer using 1-cm path length quartz cuvettes. The CYP124holoenzyme was quantified by using the Soret band by subtracting the signalof the ferrous-deoxy form of CYP124 from the ferrous CO-bound enzymegenerated with 1 mM sodium dithionite. An extinction coefficient (450–490nm) of 91 mM�1cm�1 was used (56). CYP124 ligands were assessed by using adual-beam spectrophotometer, and difference spectra were recorded at 25 °Cbetween 350 and 500 nm. Compounds that induced typical Type-I or Type-IIdifference spectra were titrated to establish their affinities toward the en-zyme (7).

CYP124 Catalytic Assays. Catalytic assays were carried out at 25 °C in glass tubesin a volume of 0.5 mL. CYP124 (120 pmol) was preincubated with substrates in50 mM potassium phosphate (pH 7.4) containing spinach ferredoxin andspinach ferredoxin-NADP� reductase. All reactions contained 10 �g/mL cata-lase and an NADPH-regenerating system consisting of glucose 6-phosphatedehydrogenase (2.4 units) and 1 mM glucose-6-phosphate. Reactions werestarted by adding 0.5 mM NADP�, control reactions contained all of thecomponents except CYP124 or NAPDH, and reactions were quenched byadding 1 mL of 1 M HCl, 5 ml methyl tert-butyl ether (MTBE), mixed, andcentrifuged (4 °C at 1,500 � g for 15 min). The organic phases were evaporatedand converted to the respective trimethylsilyl (TMS) derivatives by resuspen-sion in 50 �L of BSTFA (Pierce) for 2 h at 25 °C. Reaction mixtures wereanalyzed by using an HP5790 gas chromatography system fitted with aDB5-MS column (30 m � 0.25 mm � 0.25 �m) as described in ref. 57. Separationwas achieved by using a column temperature of 70 °C for 1 min, increased by10 °C/min up to 300 °C, and finally held at 300 °C for 1 min. Specific activitieswere determined at saturating concentrations of substrate, typically 5 � Km.

Crystallization, Data Collection, and Crystal Structure Determination. The initialscreening of crystallization conditions was performed by using commercialhigh-throughput screening kits (Hampton Research), a nanoliter drop-settingMosquito robot (TTP; Labtech) operating with 96-well plates, and a hanging-drop crystallization protocol. Optimization of conditions was carried outmanually in 24-well plates. The protein was from 0.5 mM frozen stock in 50mM potassium phosphate (pH 7.5), and before crystallization, the protein wasdiluted to 0.1–0.2 mM by mixing with 10 mM Tris�HCl (pH 7.5) alone orsupplemented with 2 mM phytanic acid. Crystals of the ligand-free CYP124grew from 1% PEG MME 2000, 0.1 M Na cacodylate (pH 6.8), and 0.9 M succinicacid. Crystals of the CYP124-phytanic acid complex grew from 21% PEG 4000,0.1 M Bis-Tris (pH 5.5) and 0.2 M Ca acetate. Before data collection, the crystalswere cryoprotected by plunging them into a drop of reservoir solution sup-plemented with 20% glycerol and flash-frozen in liquid nitrogen. Diffractiondata were collected at 100–110 K at beamline 8.3.1, Advanced Light Source,Lawrence Berkeley National Laboratory, Berkeley, CA. Data indexing, inte-gration, and scaling were conducted by using MOSFLM (58), HKL2000 (59), andELVES (60) software suites. Crystal structures were determined by molecularreplacement using the atomic coordinates of Mtb CYP125 as a search model(Table 1).

ACKNOWLEDGMENTS. We thank Andrew Munro and David Leys for provid-ing the CYP125 coordinates before publication, Sylvie Kandel for CYP2E1,

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Hugues Ouellet for helpful advice, Chiung-Kuang Chen and the staff membersof beamline 8.3.1, James Holton, George Meigs, and Jane Tanamachi (theAdvanced Light Source at Lawrence Berkeley National Laboratory) for assis-tance with data collection. This work was supported by National Institutes of

Health Grants grants RO1AI07824 (to P.R.O.d.M.) and RO1GM078553 (toL.M.P.). The Advanced Light Source is supported by the Director, Office ofScience, Office of Basic Energy Sciences, of the U.S. Department of Energyunder Contract No. DE-AC02-05CH11231.

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