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William B. Jakoby and J. V. Bhat 1958. MICROBIAL METABOLISM OF

OXALIC ACID. Microbiol. Mol. Biol. mmbr.ASM.ORG - Rev. 22(2):75-80.

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MICROBIAL METABOLISM OF OXALIC ACID WILLIAM B. JAKOBYl AND J. V. BHAT2 Although the synthesis of urea by Wbhler in study of compounds supporting bacterial growth, 1828 is credited with dispelling the notion of Den Dooren de Jong (7) and Ayers et al. (8) vital forces involved in the synthesis of organic were unable to obtain growth of a variety of chemicals, the first to be as- saprophytic bacteria when was the sole mmbr.ASM.ORG - sembled from inorganic matter is the subject of carbon source. Much of the literature pertaining the present review. Legend has it that because to attempts at demonstrating bacterial utiliza- of the empirical formula, written as C203 H20 tion of oxalate has been reviewed (9). at the time, oxalic acid prepared from To date several organisms have been isolated by W6hler in 1824 was judged to be "inorganic." which are able to grow on a medium containing More pertinent here is the interest of the micro- only inorganic salts, a nitrogen source, and biologist and biochemist in oxalic acid as the oxalate. These strains have been obtained by July 19, 2010 most highly oxidized of organic compounds. the enrichment culture technique from a variety Oxalate enjoys ubiquitous distribution in the of sources and their routine isolation has been animal and plant kingdoms. In humans, oxalic simplified by a technical modification of the en- acid and its salts are present in urine, in blood richment medium (10). Of the organis ob- to the extent of 4 mg per cent (1), and in kidney tained, only the following have been charac- "stones", of which they may form a major con- terized to the extent of allowing taxonomic stituent (46). Although minute amounts are syn- classification: Pseumonas astragali (11), P8eudo- thesized by mammals (2, 3) it is reasonable to monas oxalaticus (12), Pseudomona rimaefaciens suppose that much of the oxalate of animals (13), Pseudomonas extorquens3 (14), Vibrio originates from the oxalate ingested with plant oxaliticus (10), Bacterium oxaliticum (17), and material. Enormous quantities of oxalate are often Mycobaderium lacticola (18). Other isolated found in leaves (4), amounting in one case to 20 organisms include unidentified coryneform bac- per cent of the dry weight of the plant (4). Accu- teria (19), pseudomonads (19-21), and several mulation of oxalate by fungi, particularly by saprophytic mycobacteria (22). Several species species of Aspergilue, Penicilium, and Mucor of Nocardia (23, 24) and Streptomyces (23, 25) (5), are of such an order that they may be were able to utilize oxalate as the sole carbon adapted to industrial fermentation for this com- source. Other Streptomyces required supplemen- pound.soreOte tetryereurduplmn AounimaAnimals appearaper noto too cataboliseaaolz xaae tation with yeast extract before oxalate utiliza- appreciably (2, 3, 6) and, although higheroxalatetinwsdmsraeplants tion was demonstrated (26).2) as well as mosses do metabolize it to some ex- It is of interest that attempts to isolate anaer- tent, it would seem that a major portion Of obes utilizing oxalate have been unsuccessful oxalate scavenging is left to the bacteria and, (11, 21) although it has been reported (27) that possibly, the fungi. Oxalate, however, does not Pseudomona aeruginosa will grow on oxalate generally accumulate in fertile soil to the extent under anaerobic conditions. that its toxic properties (i.e., it is a powerful chelating agent) interfere with plant growth. OXAIC ACID CATABOLISM However, the organisms generally available in Soluble enzyme systems have been demon- laboratory culture collections are not proficient strated which catabolize oxalic acid by at least in oxalate oxidation. Indeed, in an exhaustive ' The isolation of Bacillus extorquens by Bassalik 1 National Institute of Arthritis and Metabolic (14) represented the first report of an organism Diseases, and National Institute of Dental Re- able to utilize oxalate as the sole carbon source. search, National Institutes of Health, Public Although this strain perished, Janota (15, 16) has Health Service, U. S. Department of Health, been able to isolate an organism resembling the Education and Welfare, Bethesda, Md. original in every respect, which she has preferred 2 Fermentation Technology Laboratory, Indian to name Pseudomonas extorquerm because of its Institute of Science, Bangalore, India. characteristic morphology. 75 76 JAKOBY AND BHAT [VOL. 22 three routes and involve oxidation, decarboxyla- oxalate is thought to be the system active in the DOWNLOADED FROM tion, or activation followed by decarboxylation. white-rot, wood-destroying fungi (36). The Oxidation. The oxidation of oxalic acid, re- categories of white-rot and brown-rot fungi were sulting in the formation of carbon dioxide and found to be distinguishable by the accumulation hydrogen peroxide (reaction 1) has been found of oxalate by the latter group and the ability to in mosses and in higher plants. Within this catabolize oxalate by the former (36). The puri- limitation a wide spectrum of species fied decarboxylase from Collyvia veltipes (37) is a COOH stable enzyme, specific for oxalic acid decarbox- (1) + 02 2CO2 + H202 ylation in the reaction shown below: mmbr.ASM.ORG - COOH~~~~~~~~8 HOOO+CO(02) is capable of carrying out the reaction (28). (3) GO HCOOH + C02 Peroxide has been identified as the product (29) COOH and the stoichiometry of the reaction was es- The enzyme is peculiar in that catalytic tablished (30). More recently this work has been quantities of oxygen are required for the reac-

confirmed with extracts of (31) and of tion to proceed although the over-all process does July 19, 2010 several mosses (32). This type of reaction is con- not utilize oxygen stoichiometrically. Oxygen sistent with the suspicion of flavoprotein media- can not be replaced by a variety of compounds, tion. Although the moss enzyme has been found including hydrogen peroxide, quinone, cyto- to be markedly stimulated by FMN4 or ribo- chrome c, or the flavin and pyridine nucleotides. flavin but not by FAD (32), rigorous proof of Activation and decarboxylation. The only bac- flavin participation as a coenzyme is not avail- terial system described for the utilization of able. oxalate was found in a pseudomonad and can, Consideration should also be given to the in the presence of acetyl-CoA and ThPP, catalyze possibility of a pyridine nucleotide-linked oxida- the conversion described by reaction 7 (20). The tion of oxalic acid (reaction 2). Convincing evidence presented suggests that reaction 7 is evidence for the existence of such a dehydrogenase the summation of reactions 4 to 6. is not available. However, mention oxalate + acetyl-GoA oxalyl-GoA COOH '4' + acetate (2) + DPN+(TPN+)-+ 2CO2 (ThPP) GOGH (6) oxalyl-CoA - (formyl-CoA) + DPN(TPNH) + H+ + CO2 is made of the demonstration (33, 34) of the (6) (formyl-CoA) + H20 formate + CoA reduction of methylene blue when oxalate and (7) oxalate + acetyl-CoA plant extracts were incubated anaerobically, a (ThPP) result which could be interpreted as due to the + H20 - formate involvement of pyridine nucleotide or of flavin + CO2 + acetate + CoA coenzymes. In the case of barley preparations Pyridine nucleotides are not directly involved (34) the addition of a yeast kochsaft was found in oxalate decarboxylation. A DPN-specific to be necessary for this activity. In one case formic dehydrogenase (reaction 8) has, however, aerobic oxidation of oxalate with a moss prepa- been purified ration in the presence of cyanide was not found to yield peroxide although the participation of (8) HCOOH + DPN+ --CO2 + DPNH + H+ catalase was not completely ruled out (35). from the same organism (38) and when added to Decarboxylation. The direct decarboxylation of the oxalate system together with DPN will 4The following abbreviations are employed: produce two moles of carbon dioxide per mole of FMN, flavin mononucleotide; FAD, flavin ade- oxalate utilized. It is of interest that preliminary nine dinucleotide; DPN and TPN, di- and tri- experiments5 indicate similar requirements for phosphopyridine nucleotides, respectively; DPNH oxalate decarboxylation when Neurospora crassa and TPNH, the reduced forms of DPN and TPN, or baker's yeast preparations are used. respectively; ThPP, thiaminpyrophosphate; for- myl-CoA, acetyl-CoA, oxalyl-CoA, etc., the CoA 6 Unpublished results of Y. Yamamura, 0. derivatives of the respective . Hayaishi, and W. B. Jakoby. 19581 MICROBIAL METABOLISM OF OXALIC ACID 77 DOWNLOADED FROM OXAIC ACID FORMATION by other organisms. Nor should the possibility of other than Sincetheinitialstudies of We e with cleavage by reagents water, i.e., Asperfllu8bSince theinitial39niger (39),studesformaithe formationof of oxalicwith phosphate or CoA, be neglected. Indeed an acid has been assiduously investigated. Becausedecid(4)wchsatventefominenzyme, thiolase, from a pseudomonad has been of the large quantities of oxalate accumulated and the availability of methods for their growth and scission of 3l-ketoadipyl-CoA to form suc- under controlled conditions, the fungi have been cinyl-CoA and acetyl-CoA (reactions 12 and 13). the experimental organisms of choice. The litera- mmbr.ASM.ORG - ture beaing on oxalic acid formation durin (12) f3-ketoadipate + succinyl-COA - growth or with mycelial preparations has been ft-ketoadipyl-CoA + succinate summarized in detail by Foster (5) and by Nord (18) j3-ketoadipyl-CoA + CoA (40). Examination of the available information succinyl-CoA + acetyl-CoA indicates that a great variety of carbon sources is able to give rise to oxalate. Although such (14) oxaloacetate + succinyl-CoA -. data can only be suggestive, this work has led oxaloacetyl-CoA + succinate July 19, 2010 to the proposal of what seem to be two logical pathways for oxalate formation. Reaction 9 (15) oxaloacetyl-CoA + CoA involves the cleavage of while oxalyl-CoA + acetyl-CoA reaction 10 depends upon the Indirect spectrophotometric evidence indi- COOH COOH cated that the substitution of oxaloacetate for I fl-ketoadipate may give rise to a similar series of 100 + H20 COOR reactions (reactions 14 and 16) although neither (9) CH2 the products, i.e., oxalyl-CoA and acetyl-CoA, CH3 nor the intermediate, i.e., oxaloacetyl-CoA, have COOH been isolated from such a reaction mixture (43). COOH That a compound such as oxalyl-CoA can be formed by a transfer reaction has already been COOH COOH described for another pseudomonad (reaction 4). (10) + M 02 Formation by oxidation. Using relatively crude CHO COOH extracts from tobacco leaves, it has been demon- oxidation of . strated (44) that oxalic acid is formed from Formation by cleavage. Reaction 9, which had glyoxylic acid under aerobic conditions. The been previously proposed (41), seems to be mechanism of the reaction is not as yet clear operative in extracts obtained from A. niger (42). because of the heterogeneous protein extract A soluble preparation from this organism pro- employed but may be similar to duced equimolar quantities of oxalate and ace- tate per mole of oxaloacetate utilized. Magnesium COOH COOH ions stimulate the reaction somewhat. Under (16) + 02 + H202 these conditions the substitution of oxalosuc- CHO COOH cinate for oxaloacetate did not produce oxalate. A hydrolytic reaction with oxalosuccinate as one present in spinach. The latter enzyme (45), substrate (reaction 11) should not, however, be a flavoprotein, is active on two a-hydroxy acids, dismissed as a possible means of oxalic acid glycolic and lactic acids, with the formation of production glyoxylic and pyruvic acids, respectively. This specificity need not, of course, apply to the to- COCOOH COOH bacco leaf extracts, which are active with gly- | COOH oxylic, glycolic, and lactic acids. However, there CHCOOH + H20 CH2 is no evidence that one enzyme has the specificity (11) COOH + SI| vdneta n ezm a h pcfct CH2COOH CH2 to handle all three substrates. Attempts have been made to obtain systems COOH catalyzing the general reaction described by 78 JAKOBY AND BHAT [voL. 22 DOWNLOADED FROM equation 17, i.e., pyridine nucleotide- may be considered as playing the above described role. However, a system for extensive CO2 fixa- COOH tion by this organism has not been excluded as

(17) (OH2)5 + DPN+ + HO a possible anabolic pathway. I Plants, on the other hand, appear to handle 011 oxalate by oxidation to C02. Obviously, plant 0001 life is well adapted to C02 fixation and the oxidation of oxalate may only be in the nature

I mmbr.ASM.ORG - (CH2)5 + DPNH + Ho of a "detoxification" reaction. I 0O001 REFERENCES enzymes active in the dehydrogenation of semi- 1. ENRICO, L. 1953 Ricambio dell'acido ossa- . Such enzymes have recently been lico nei basedowiani prima e dopo intervento chirurgico. Arch. sci. med., 95, 112-130. obtained from P. fluorescen (47),( mammalian 2. CURTIN, C. O'H., AND KING, C. G. 1955 obtained July 19, 2010 brain (48), andP.dbaor'yeascbaker's yeast.. Although the The metabolism of ascorbic acid-i-C14 and microbial systems are active with succinic semi- oxalic acid-i-C1' in the rat. J. Biol. Chem. where, n = 2, glyoxylate (n = 0) is 216, 539-548. not utilized. The brain enzyme does use glyoxyl- 3. WEINHOUSE, S., AND FRIEDMANN, B. 1951 ate as well as other semialdehydes with the con- Metabolism of labeled 2-carbon acids in the comitant formation of DPNH. Oxalate, the ex- intact rat. J. Biol. Chem., 191, 707-717. pected product of brain semialdehyde dehy- 4. THIMANN, K. V., AND BONNER, W. D., JR. drogenase action with glyoxylate as substrate, 1950 metabolism. Ann. Rev. has not yet been isolated and characterized. Plant Physiol., 1, 75-108. 5. FOSTER, J. W. 1949 Chemical activities of DISCUSSION the fungi. Academic Press, Inc., New York. 6. BERNARD, K. 1954 Isotope als Indikatoren The high state of oxidation of oxalate, re- zur Erforschung des Lipidstoffwechsels. quiring the removal of only two electrons for Schweiz. med. Wochschr., 84, 506-508. C02 formation, has led to the suggestion (10) of 7. DEN DOOREN DE JONG, L. E. 1926 Bijdrage the possible "autotrophic" nature of organisms tot de kennis van het Mineralisatieproces. able to utilize oxalate as the sole carbon and Dissertation, Delft. energy source. Oxalate would be degraded to 8. AYERS, S. H., Rupp, P., AND JOHNSON, W. T. C02, thus furnishing energy which would in 1919 A study of the alkali forming bacteria turn be used for C02 reduction. With the finding found in milk. U. S. Dept. Agr. Bull. 782, that formate is an intermediate in oxalate oxida- 9. BAT . V AND KHAATA, S R 1955 tion by a pseudomonad another explanation be- Microbial decomposition of oxalate. Soc. comes plausible. It has been shown that formate Biol. Chemists, India. Souvenir, 186-190. can be oxidized with the formation of DPNH 10. BHAT, J. V., AND BARKER, H. A. 1948 Stud- (reaction 8). DPNH, in turn, may be used by ies on a new oxalate-decomposing bac- the organism as the energy source for adenosine terium, Vibrio oxaliticus. J. Bacteriol., triphosphate formation or more directly for the 55, 359-368. reduction of another mole of formate. In effect, 11. KHORSHED, M., PAVRI, M., AND BHAT, J. V. on the asso- it is suggested that the pseudomonad utilizes ciated1953 Studieswith the leavesmicroorganismsof Pongamia glabra oxalateotbybydecarboxylation.eotollowedfo..owedby the (1a- vent. Proc. Indian Acad. Sci., Sect. B, 38, mutation of formate. It would, then, be formate 171-178. rather than oxalate which serves the organism as 12. KHAMBATA, S. R., AND BHAT, J. V. 1953 both carbon and energy source. In the case of Studies on a new oxalate-decomposing bac- V. oxalitic, which cannot grow with formate terium, Pseudomonas oxalaticus. J. Bac- as the sole carbon and energy source (10), an teriol., 68, 505-507. activated formate, as suggested by reaction 6, 13. BREED, R. S., MURRAY, E. G. D., AND HITCH- ENS, A. P. 1948 Bergey's manual of deter- 6Unpublished results of W. B. Jakoby and E. minative bacteriology. 6th ed., p. 123. Wil- M. Scott. liams & Wilkins Co., Baltimore. 1958] MICROBIAL METABOLISM OF OXALIC ACID 79 DOWNLOADED FROM 14. BASSALIK, K. 1913 tUber die Verarbeitung 30. FRANKE, W., AND HASSE, K. 1937 Zur der Oxalsaure durch Bacillus extorquens n. Biologischen Oxydation der Oxalsiure. Z. sp. Jahrb. wiss. Botan., 53, 255-302. physiol. Chem., 249, 231-255. 15. JANOTA, L. 1950 Przebieg zuzywania kwasu 31. FINKLE, B. J., AND ARNON, D. I. 1954 Oxi- s zczawowego przez Pseudomonas extor- dative decarboxylation of oxalic acid. quens Bassalik, w zaleznosci od poczatkoweg Physiol. Plantarum, 7, 614-624. licesby komorek. Med. Doiwiadczalna i 32. DATTA, P. K., AND MErUsE, B. J. D. 1955 Mikrobiol., 2, 131-132. Moss oxalic acid oxidase-a flavoprotein. 16. JANOTA, L. 1957 Further researches on Biochim. et Biophys. Acta, 17, 602-603. Pseudomonas extorquens Bassalik. A micro- 33. THUNBERG, T. 1928 Zur Kenntnis der en- mmbr.ASM.ORG - organism utilizing oxalic acid. Acta Soc. zymatischen Oxydation der Oxalsaure durch Botan. Polon., 26, 207-214. Pflanzensamen. Skand. Arch. Physiol., 54, 17. KHAMBATA, S. R., AND BHAT, J. V. 1953 6-16. Bacterium oxaliticum, a new oxalate-decom- 34. FODOR, A., AND FRANKENTHAL, L. 1930 posing bacterium isolated from the intes- tber das Dehydrierungsvermogen von tine of earthworms. Proc. Indian Acad. Getreidesames in Anwesenheit von Pflan-

Sci., Sect. B, 38, 157-160. zensaure und Purinsubstanzen als Wasser- July 19, 2010 18. KHAMBATA, S. R., AND BRAT, J. V. 1955 De- stoffdonatoren. Biochem. Z., 225, 417-425. composition of oxalate by Mycobacterium 35. NIEKERK-BLOM, C. J. 1946 Notes on the lacticola isolated from the intestine of earth- oxidation of oxalic acid by mosses. Proc. worms. J. Bacteriol., 69, 227-228. Ned. Akad. v. Wetensch., Amsterdam, 49, 19. KHAMBATA, S. R. 1954 Studies on some 1096-1100. associated with the intestine 36. SHIMAZONO, H. 1955 Oxalic acid decarbox- of the Indian earthworm. Dissertation, Uni- ylase, a new enzyme from the mycelium of versity of Bombay. wood destroying fungi. J. Biochem., Ja- 20. JAKOBY, W. B., OHMURA, E. K., AND HAY- pan, 42, 321-340. AISHI, 0. 1956 Enzymatic decarboxyla- 37. SHIMAZONO, H., AND HAYAIsmH, 0. 1957 tion of oxalic acid. J. Biol. Chem., 222, Enzymatic decarboxylation of oxalic acid. 435-446. J. Biol. Chem., 227, 151-159. 21. JAYAsURIYA, G. C. N. 1955 The isolation 38. JAKOBY, W. B. 1956 Enzymatic decarbox- and characteristics of an oxalate-decom- ylation of oxalic acid. Federation Proc., posing organism. J. Gen. Microbiol., 12, 15, 282. 419-428. 39. WEHMER, C. 1911 Chemical activity of the 22. LONG, E. R. 1922 The nutrition of acid- Aspergillaceae. In Technical mycology, Vol. fast bacteria. Am. Rev. Tuberc., 5, 857- 2, pp. 259-277. Edited by F. Lafar. Charles 869. Griffin and Co., Ltd., London, England. 23. MCCLUNG, N. M. 1954 The utilization of 40. NoRD, F. F., AND WEISS, S. 1951 Yeast and by Nocardia species. J. mold fermentations. In The enzymes, Vol. Bacteriol., 68, 231-236. 2, Part 1, pp. 684-790. Edited by J. B. Sum- 24. MULLER, H. 1950 Oxalsaure als Kohlen- ner, and K. MyrbAck. Academic Press, stoffquelle fur Mikroorganismen. Arch. Inc., New York. Mikrobiol., 15, 137-148. 41. LYNEN, F., AND LYNEN, F. 1948 Die bil- 25. STARKA, J. 1955 Rozkad oxalatu pudnimi dung von Oxalsiure durch Aspergillus niger. mikroorganismy. Preslia, 27, 21-26. Ann., 560, 149-162. 26. KHAMBATA, S. R., AND BRAT, J. V. 1954 42. HAYAISHI, O., SHIMAZONO, H., KATAGIRI, M., Decomposition of oxalate by Streptomyces. AND SAITo, Y. 1956 Enzymatic formation Nature, 174, 696-697. of oxalate and acetate from oxaloacetate. 27. RoBINSON, G. L. 1932 The growth of B. J. Am. Chem. Soc., 78, 5126. pyocyaneus in synthetic media. Brit. J. 43. KATAGIRI, M., AND HAYAIsHI, 0. 1957 En- Exptl. Pathol., 13, 310-317. zymatic degradation of i3-ketoadipic acid. 28. HOUGET, S., MAYER, A., AND PLANTEFOL, L. J. Biol. Chem., 226, 439-448. 1928 Sur une forme particuliere d'oxyda- 44. KENTEN, R. H., AND MANN, P. J. G. 1952 tion biologique. II. Ann. physiol. physico- Hydrogen peroxide formation in oxydations chim. biol., 4, 123-128. catalysed by plant a-hydroxyacid oxidase. 29. HOUGET, J., MAYER, A., AND PLANTEFOL, L. Biochem. J., 52, 130-134. 1927 Sur une particuliere d'oxydation bio- 45. ZELITCH, I., AND OCHOA, S. 1953 Glycolic logique. Compt. rend., 185, 304-306. acid oxidase. J. Biol. Chem., 201, 707-718. 80 JAKOBY AND BHAT [VOL. 22 DOWNLOADED FROM 46. ARCHER, H. E., DORMER, A. E., SCOWEN, E. F., transaminase and semialdehyde dehydro- AND WATTS, R. W. E. 1958 The aetiology genase. Science, in press. of primary hyperoxaluria. Brit. Med. J., 48. ALBERS, R. W., AND SALVADOR, R. A. 1958 I, 175-181. Succinic semialdehyde oxidation by a sol- 47. SCOTT, E. M., AND JAKOBY, W. B. 1958 Pyr- uble dehydrogenase from brain. Science, rolidine metabolism: Soluble y-aminobutyric in press. mmbr.ASM.ORG - July 19, 2010