' SEP-224% TUE 03: 02 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 .P, 02/15 ' . SEP 25 '98 B9:0?cIM WC MICRO & IMMUN 606 257 8994

' .P

' Edvard DeMell P.6/13

Pregress Rkport The Role or Purine Degradation i n Methane Biosynthesis

and Eaergy Production i n MeChanococcuG vsnnielif

m 3 z m i nathwev in mth -

described for rep*, The

H. uannielfi pathway differs in a few ZeSpects from the Clostridial pathway. The pathway of Clostr idia uses tetrahydrofolic acid (TICIF), whereas the pathway of M.'vannieli i uses tettahydromethan- opterin (H,HPt) as a cofactor Sn the transfer OX both the fomnimino moiepy of fonaimincglycine and apparently in the cleavage of gly- cine by a glycfna decarboxylase typo mechanism that i s dwpendent upon a t least H,KPt and either NAD* o r NADP' (both seem t o work in crude Known qlycipe decarboxylases catalyze the fol- Lowing reaction:

We have developed an assay for this rtkctlion in the M. vanniel i i system baoea on the relaase of COa from L-[l-14CIglycine. We plan to purify this enzyme complex so that .we may better. characterize the involvement of H l M P t . We a lso have developed assays for the ffrst two enzymes of this pathway w i t h an eye towrxd t b i r purifi- cation and characterizarion. If tb&s pathway i s regulated, we would expect the regulation to occur a t this step, hence our in- terest in this enzyme. The assay for xanthine amidohydrolase (Figure 2, E3) employs C-18 reverse phase separation Og xanthine from the pzoduct o f the reaction, 4-ure~do-5-im~dazolecarboxylic acid, This product is readily identifiable by its charaotecist~c pH-depcnd@nt W spectrum, The assay for the reaction catalyzed by the next e n z m (Figure 3, E4) I s based on the release o f ''CO2 from [2-"C J 4-uw~de-5-f~da~olecarboryLfc acid prepared enzymatically from f2-L'Clxanchine. We with to study this enzymm because of i t s intttesting enzymology. We have detenq.lned tha t the enzyme f f o m M. v a n n f e l i i requires or reqa fQr acti$tity. !rhe s i t e of cfeavage, the ureide moiety and the requirement for a divalent transition metal are reminescent o f ureaset which has N f q Z as a component o f its active site. We have waited until we have developed assays for all three of theae enzymes in ordet that we may purify them concurrently, One of 6ur bottlenecks I s pzoduction o f cellular material, which is also the only soutc.6 of methanogen coenzymes. This pZan w i l l save our valuble supply of cells,

2) 01af: $P o f p e dearadati on to methanaue nesis . The '

ciegrieatfoa of-thsne b=. vanrriel i i produces f ive cI units, The C-2, C-4 lvra glycine), and C-6 atoms O f xanthine are liberated as CO,, and the C-5 (via glycine) and C-8 atoms are transferted to Hpt, since our initial studles have been pexformed with crude extracts, we cannot be absolutely $Ute what state of is

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or use- fulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any spc- cific commercial product, proass, or service by trade name, trademark, manufac- turer, or otherwise does not necessarily constitute or imply its endorsement, ncom- mendation. or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

DISC LA1 M ER

Portions of this document may be illegible in electronic image products. Images are produced from the best available original document.

SEP-22-9l3 TUE 03: 02 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 P, 03/15 ', SEp 21 '98 m:@?RW UK MICRO & I m m6 257 8994 Edward DeMoll P.7113

5

formed on transfer ~f the carbon atam$ (due to interconvcrsSons of the oarsous fo- of the coZactor catalyzed by enzymes in clue crude extract), but based on analogous reactions i t is l i k e l y t h a t C-5 of xanthine (C-2 of glycine) and H@t generate sV5 lV%hethylene-H,MI?t, and that C-8 of xanthine and X&Pt generate Nk, t#@+ethenyl-H,Wt. since both of these cofactor forms are also components of the meth- ane biosynthetic pathway or are zequised i t r O t h e r biosynthetic steps (such ad TMI? synthesis from d W ) , It was reasonable t o as- atme that a11 of chase carbon atoms may enter the methane biosyn- thetic pathway or may be used to synzhesize cellular material, We have found Wat the former case is true by di tect evidence, and we h o w that the latter 56 true because $'C from in various gosit ions in xanthine and glycine are incorporatedinto acid fnsolublemater- Sal w i t h t i m e . We have yet t o assess the extent; t o which purines Ean Contribute carbon for growth and methanogmesis. The main limitation t o thls appears t o be their limited solubflity, as= pech l ly a t neutral pX. We did not appreciate t h i s i n i t i a l l y because M. vannie l i i grows vezy w e l l with formate as a carbon source i n the pw range of a - 0 t o 0.8, Also there is, not surpris- ingly, a temperature effect on the solubility of these compounds, however this temperature effect i s not as pronounced i n going from, for example 25 to 4OoC, as the substantial increwa in solubility of xanthine, guanine, or hypoxanthine seen when going from pH. 7 .O t o 8 -5 , We considered that there might be a growth retriction due to putihe insolubility when we had problems trying to grow JY. van- nielli a t 3OoC with uric acid as sole nitrogeh source and COJ& as carbon and energy source a t pfi 7 , O , This growth restriction As due t o the Sact that most purines and their raspective nucleoside$ have pKrs at sligitly basic pH (xanthine and xanthosine, 7.4; guanine and guanosine, 9.0; hypoxanthine and inosing, 6 .9 ) . This means thar; M, vannieJrti growing with formate as carbon source is better

* suited for utilizing pu;rhes and nucleosides than is a micro- orgmism which grows best near pH 7.0. Increasing the pH t o 8.5 doee not aubsfantially affect the solubility of uric acid (pK, llr3), hawaver. For optimal growth of micr0OrganlskaS in the l'aboratory with uric acid, a chemical trick must be applied,

Solubilizing uric acid has evidently been the source of sev- eral of our problems. We have sotten inconsistent results when we have t r i e d to grow M, vannleldd with uric acid as sols pitrogen source. Sometimes we would get ne growth, other times M. vannielii would grow t i n e , Different bacches of c a l l s would have varying levels of xanthine dehydrogenase, tha enzyme necessary for uric acid uti l izat ion, that is induced by the presence of some as yet undetermined le-1 af uric acid. We have resolved the problems of uric acid insolubility, A look at past literature dealing with growth of other anaszobes on uric acid indicates t h a t others must hove.accasianally had solubility problems, but they usually solved the problem empirically, and were aided by the nore rap&d growth of their bactrria, We found that the t r i c k t;o growing bacteria on uric acid i s to insuse that the rnedfium is supersaturated with uric acid when inoculated. When the growth medium is put together, uric acid (2 to 5 mM) 1s added first, the medium i s heated, and NaOH is added until the purine dissolves. Other m e d i u m oamponents are added, the pH is lowered to approximately 8.0 t o 8.5, and the

- SEP-22-94 TUE 03:02 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 P, 04/15 PI 8/13 Edward DeMoll

medium is cooled to around 40T. A t this point, uric acid is supersaturating. ' If inoculation with the microorgani~m i s done at this point, uric acid can evidently be metabolized so that i t may be u$ed as sole nitrogen source for growth. During the next f e w days uric acid solubility goes toward the equilibrium condition and the compound falls out of solution. However, this has usually been a long enough time to allow M. vanrrlelil t o use the compound as a source of nitrogen. Since tables of purine solubilities that were available t o us are not complete with xespect t o all temperatures and pH values of interest, we carried out solubility tests o f vari- ous purines at different temperatures and pH values in OUL standard M. vanniel i i granth medium to confitm our susp$cions that the pK and temperature dependent solubility problems o f growth on purines might account ,€or the variability in growth we had been seeing. Our results supported the conclusions made i n the above discussion. Additionally our results may explain why methanogens that grow at mesophilic temperatures near pH 7, and that can utilize purines as nutrients at lower concentrations (approximately 0.1 M), might not ever encounter purines a t levels above 1 lnM where M, vannielii i s able t.0 used them as a general source o f nitrogen,

Purine and purine nucle6side utilization .-by. M, v a n n i e z i i . We have found that M, vannielif does either of two things with purines it takes from i t s growth medium. IC either degrades them according to the pathway described later or it converts them t o nucleotide monophosphates which are used for a variety of cellular needs. The incorporation oE various purines Onto the purine nucleotide pool has aLso been noted fo r other methanagens. We performed sxperi- m a t s wherein M. v a n n i e l f i was grown i n the presence of 20 mM NH4* and 50 pM o f either 15'-3HJguahoshe, [U-2AC] guanosine, ut [8- 'kJguanine. The specific activities of GMP, AMP, CMP, and UMP

I i so lated from RNA, and Factor 420 ( F I L I I ) and tetrahydromethanopterh (H,MPr.) were then analyzed and compared to the specific a c t i v i t i e s of the added guanine or guanosine. Additionally, the amount of labeled compound taken into the cells was determined- 16%, 38%, and 60% of the labeled atoms that originated in [ 5 ' - 3 H 1 ~ u a ~ s i n e , W a 4 C l guanosine, and [8-'*C J guanine, respectively, were found inside the cellsI These values could be underestimates o f the a m o u t s taken up if a significant amount o f the fragile cells lysed during growth and harvest, The results f r o m specific act iv i ty tneasure- ments are summarized in T a b l e 1.

1) A t 50 pM guanosine is n o t take up as completely as guanine (see above). That less of the '€I-label than the %-Label from guanosine w a s found in the cells could be due to ex-perimental variation or due to the fact that 3H is more readily exchangeable, so it could be lo$ t to the medium as Cn, or &O. when guanosine is taken into the cell, the ribose is readily cleaved from guanine. When the label is only in ribose (Sr-3~-label), one sees that the specific activity of GMP fs 1/12 of that when the label i s in both the ribose and guanine (U-i4C-labeJ.) . This difference i s not accounted for by the uptake/exchange difference in these two compounds, which was 16% vs. 388, a ratio of 1/2 .4- 3) Although some label is Eound in all nucleotides in each of these experiments, there is no significant conversion o f guanine, guanosine, or GMP to At@. This

Several conclusion$ can be made from these results.

2)

SEPA22-9& TUE 03:03 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 * , !SW 21 '98 E19:OSFIM UK MICRO 8 T W 257 8994 Edward DeMOlL P.9/13

is particularly evident in the [8-%1 guanine study. The added SO ps guanine acqounts for 71% o f the 6MP isolated f rom RNA. ?his indicates, that hl. uannislii takes up guanine avidly, and that de novo guanmna nucleotide synthesis has bean 8ignificantly inhibited os Fepresaed by the guanine thar was transported, 5 ) Synthasfs of H,MPt and E''pe from guanine nuc;leotidts with loss o f the 8-carbon (at least in the case of R p t ) is affirmed. 6) Anunoniwn ion at a concentration o f .20 mM does not inhibit guanine (10 to SO pa) uptake. 7) No conclusive statement can be made about the amount o f guan4nr degradation that occurred i n these e x p e r h n t s , because CQ, and CHI, which should be the main depositories f o r Jabel, were not analyzed, 8) We cannot explain the relat5vely high specific ac t iv i ty of the Fato from the I8-%1 guanine experiments, however this Value i s close to the 35 valua obtained by previous invea- tigatoro (Jacnchan, et a i , 1984. Arch. Microblol. 13?:362-365).

4 )

Table 1

[ S ' 0% 1 G-R KU-"C] G-R [8-"Cl G (3.20 x 3 0 9 (1.84 x loa2) (2 .78 x 10")

CMP

AMP

GMP

4 .9 x l o g 10.10)

-5.3 x 103 (0.10)

4.7 x 109 (0.09)

P, 05/15

4 - 1 ' x 10' (0.22)

4.6 x 10' (0.25)

2.9 x 10' (0.01)

5.7 x 109 (0.201

n4m t 1.2 x lolo (0.24) 2.1 x l o l l (12)

2-0 x 10" (71)

1.3 x loxr (4.61

8-1 x 10' (0.29)

.The values a t the top-of the table are the compounds ( w i t h rhefc sp. acc, below) added t o culturds o f M. vanniell i . The values in the table are the sp. act. of che various compounds l i s ted at the left, which were purified from the cells hasvested fzom the cultures and analyzed. The number in pasentbeses next t o the sp. act . is the percent of the sp, act- of the compound at left cempared co the sp. act. o f the added compound.

Xanthine D W d r aaenase. This enzyme i s responsibla for the intezconversions, through anaerobic oxidations or: reductions, o f certain purines. We have hown for aeveral years that Che enzyme from M, vannieli i can u t i l i z e either uric acidl xantbine, ox hypoxanthine as a substrate. We purified this enzyme some t imer ago, but had only- a partially charactcrlzed it due to a lack of material. Purification of additional quantities i n the interim has been thwarted by a lack o f suitable {with high enough specific activity) ce l lu lat material, tha absence of the previously used

' .SEP-22-B TUE 03:03 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 .P, 06/15 SEP 21 '98 09:OS~ UK MICRO & IMMLM 606 257 8994 Edward D@Moll PI 1043

HpLC system, and the desire to perrorm certain studies which re- quire a relatively large quantity of enzyme, f o r example the char- acterization of the form o f molybdenum cofactor present i n the enzyme. Our characterizations o f the enzyme have shown that it has three kinds of submits of approximate molecular weights 91,200, 34,700, and 15,900, Basad on native polyacrylamide gels, which indicate a molecular weight of approximately 165,000, the subunit conposition is estimated to be either 1:2:1, 1:1:2, or 1:1:3,

= Efforts are ongoing t o obtain a better value for the native mole- cular weight, so that we rnay know the exact submit composition. We have n o t yet completed the metal analysis o f the enzyme, because we are currently performing k i n e t i c studies ahd do not yet have enough enzyme to incinerate in a metal determination. So that we rnay consezve enzyme, xanthine dehydrogenase from these studies i s being saved for the various analyses which require destruction of the enzyme. Uric acid induces synthesis o f this xanthine dehydro- genase (DH) fn fact the enzyme is undetectable in cells that have not been exposed to uric acid. XDH catalyzes the reversfble oxidation and reductions of uric acid to xanthine to hypoxanthine. The kinetic experiments we have done so far tend to indicate that the oxidat ion of xanthine involves a disproportionation to uric acid and hypoxanthine when there i s no other electron acceptor present I In order to complete these studies w e will evidently need t o EPLC p u r i f y out purines prier to use, since those commercially available contain various amounts of other purines.

W e decided t o see if other biochemicals other than purines and ammonia can supply the nitrogen requirement for M. vannie l i i - We have found that a mix of 17 amino acids (the usual 20 minus glutamine, asparagine, qnd cysteine) added at 50 mg/L each, can supply enough nitrogen to M. v a n n i e l i i grown In OUT standard 1.5% formate medium t o replace N&* and allow a reduced, but significant Ievel of growth as measured by the in- crease in protein in the culture, In the Course of these studPes, we also discovered that even a t high growth rates, Y. v a n s i e l i i uncouples methane synthesis from growth. When amino acids replaced ammonium ion as the nitrogen source, although the organism grew only about 1/3 as much as cultures with ammonium ion (10 mM) and only about 1 / 4 a5 much as cultures with ammonium ion and amino acids, the methane production measured in each culture was approx- imately the Same (Withih an average of 25%). Also a culture w i t h no nitrogen added (so that the only nitrogen source was the carry- over in the 10% inoculum) grew very little, only 1/10, 1/30, and 1 / 4 0 as much as the amino acids only culture, the ammonium i on only culture, and the ammonium ion plus amino acids culture, uespec- tlvely. Despite t h i s slight amount Of growth, the cqltuie still produced l / S the amount of methane as lteen in Che other three cul- tures, The no nitrogen culture had a ra t io of methane produced to growth seven t imes higher than the culture with ammonium i o n and amino acids. Further investigation found that each of the 37 amino acids alone, when actiraq as the sole nitrogen source a t 20 MM, allowed €or growth and methanogenesis. However, in these cases the rat io of methanogenesis to.growch was even higher than in the 17 amha acid mix. Apparently a l l amino acids may deaminated t o enough of an extent to a l l o w for some growth. These results com-

Nit.roczen sources of M. v a n n i e l i f .

I 6 SEP-22-98,TUE 03: 04 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 Pa 07/15

pZete oui picture 4 f how M. v e n n i e l i i adjusts its intermediary merabolism to aqcomodate i t s nitr6gen requirement.

R which realre au;LB.,M, vannie l i l . From evidence we b a m e d in our stud& of purine degradation in M. van- n i e l l i it Ls Likely that intermediary metaboli8m of C, units i n and 4ut o f the main methane biosynthetic pathway in this organism is mediatad by B,Wt. In Figure 2 some o t these reactions ace shown brlnching off o f the main pathway in much the same way that: various pethways f low i n and out of, for example, the Emdsn-Meyerhof-Parnas pathway of glucose utilizktion. We have show that reactions 3 and 5 take exist . Others have shown the existifice of feaction 6. For a time w e were interested t o 668 iZ raactions 7 ahd 8 could be car- r i e d out in M. venniell i . ff sot this would have been the first demonstration of substrate 1evel.phosphorylakfon in a rnethanogen. It would also have been the only demonstration o f W"'formyl-H,MPt, The analogous THF compoufid is well known and 1s used in these two raactions an well as in formylatlon of methianine in eubacterial protein synthesis. We have been unable t o demonatrate any ATP dependent formylation Of & M P t , and archacbacteria do not foxmylute methionine. Is there a raze for the nr"'-form of this cofactor? There is only one more known role, and we are presently checking it out. That methanbgens do not 'Carry aut the Substrace level phosphorylation reaction i 8 further evidence that more than one ATP might be derived from each methane molecule formed. This is thought t o be khe case due t o the heces- s i t y of recovering more than the on@ ATP expended te activate acetate in the case of methanogens grown with acetate as carbon source. Perhaps methanogens .do n o t utilize the substrate level phosphorylation step since the do better energetically simply by sending the C, uni t i n t o methane synthesis.

This rofe is Sn purine biosynthsis.

a... , 1 -I . - . . -

,

SEP"22-9B.TUE 03: 04 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 P, 08/15 \

Eduard DeMd II P I 12/13 S P 21 '98 09:18AM W MICRO & IMMUN 606 257 8994

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SEPA22-9&TUE 03:04 PM UK RESEARCH FOUNDATION FAX NO, 606 323 1060 P, 09/15 P . 13/13 Edward PoMolZ b . - SEP 21 ‘98 89:iBwvl LK MICRO & I W 686 257 8994

2

Protein Synthesis InlNstian in Eubacteria

4

w

w ADP

p:

A I P L