Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes

Lung, Hui-yu; Baetz, A L; Peck, Ammon B.

doi:10.1128/jb.176.8.2468-2472.1994

Cited by 73 publications

(60 citation statements)

References 21 publications

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“…formigenes expresses a unique gene required for the catabolism of oxalate (oxc, encoding oxalyl coenzyme A decarboxylase). This gene has been cloned and sequenced [14]. Sequencing of the 5k ends of oxc from numerous isolates of O. formigenes identi®ed unique, highly conserved regions which permitted the synthesis of a genus-speci®c PCR primer pair [13,17].…”

Section: Discussionmentioning

confidence: 99%

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Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples

Kwak¹,

Jeong²,

Lee

et al. 2001

BJU International

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Objective To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. Materials and methods Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-speci®c oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to con®rm oxc. Results A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the ampli®ed product was compatible with that of oxc. Conclusion O. formigenes can be identi®ed easily and ef®ciently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was signi®cantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.

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Section: Discussionmentioning

confidence: 99%

“…Therefore, O. formigenes is reportedly suitable for DNA-based identi®cation [12,13]. O. formigenes expresses a unique gene, oxc, encoding oxalyl-CoA decarboxylase used in the catabolism of oxalic acid [14]. Sidhu et al [12] established a PCR-based assay system targeting oxc for detecting this organism.…”

Section: Introductionmentioning

confidence: 99%

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples

Kwak¹,

Jeong²,

Lee

et al. 2001

BJU International

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“…Thus, the frequency of oxalate-degrading strains is likely low in this species. is a well studied organism which metabolizes oxalate using oxalyl-CoA decarboxylase and formyl-CoA transferase and produces CO, and formate as the end products at a 1:1 ratio (4,5,23). Although the oxalate-degrading mechanisms of E. faecalis remain to be clarified, oxalate-degrading E. faecalis had 3 proteins (40, 48 and 65 kDa) that were absent in non-oxalate-degrading strain.…”

Section: Discussionmentioning

confidence: 99%

Oxalate‐Degrading Enterococcus faecalis

Hokama

Honma

Toma

et al. 2000

Microbiology and Immunology

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An oxalate-degrading Enterococcus faecalis was isolated from human stools under anaerobic conditions. The bacteria required a poor nutritional environment and repeated subculturing to maintain their oxalate-degrading ability. The E. faecalis produced 3 proteins (65, 48, and 40 kDa) that were not produced by non-oxalate-degrading E. faecalis as examined by SDS-PAGE. Antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) obtained from Oxalobacter formigenes (an oxalate-degrading anaerobic bacterium in the human intestine) reacted with 2 of the proteins (65 and 48 kDa) from the E. faecalis as examined by Western blottings. This is the first report on the isolation of oxalate-degrading facultative anaerobic bacteria from humans.

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“…Expression and Purification-A BL21(DE3) Escherichia coli expression strain transformed with pET-9a carrying the wild type oxalyl-CoA decarboxylase (oxc) gene from O. formigenes (20) was generously supplied by Dr. Harmeet Sidhu (Ixion Biotechnology, Inc., Alachua, FL). Cells were grown in LB broth to an A 600 of 0.3-0.6, and protein expression was induced by the addition of isopropyl 1-thio-␤-D-galactopyranoside to a final concentration of 0.4 mM.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Berthold

Moussatche

Richards

et al. 2005

Journal of Biological Chemistry

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Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 Å resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three ␣/␤-type domains, commonly seen in this class of enzymes, and the thiamin diphosphatebinding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.Oxalic acid is one of nature's most highly oxidized organic compounds, and its dianion is a strong chelator of metal cations, especially Ca 2ϩ , causing oxalate to be highly toxic to many organisms (1). In humans, elevated levels of oxalate are associated with several diseases, including the formation of calcium oxalate stones in the kidney (urolithiasis), renal failure, cardiomyopathy, and cardiac conductance disorders (1-3). Relatively large amounts of oxalate are introduced into the body through the diet, although this diacid may also arise as a byproduct of normal cellular metabolism (4). Because humans, in common with other mammals, are not able to degrade oxalate, this compound must be eliminated by excretion in the urine or via the intestine (5). The recent observation that a symbiotic, gut-dwelling bacterium, Oxalobacter formigenes, may regulate oxalate homeostasis in humans, therefore, has important implications for efforts to develop new strategies for treating oxalate-related diseases (6). O. formigenes is an obligate anaerobe bacterium found in the gastrointestinal tracts of vertebrates, including humans, and is unusual in that it employs oxalate as the sole energy source for its survival (7,8). As a result, this bacterium not only degrades free oxalate entering the intestine lumen but also creates a transepithelial gradient favoring oxalate secretion and preventing absorption of oxalic acid in the lower tract of the intestine (9). A direct correlation between the number of recurrent kidney stone episodes and a lack of O. formigenes in the ...

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Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes

Cited by 73 publications

References 21 publications

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples

Oxalate‐Degrading Enterococcus faecalis

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

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