Characterization and heterologous expression of the oxalyl coenzyme A decarboxylase gene from Bifidobacterium lactis.
Oxalyl coenzyme A (CoA) decarboxylase (Oxc) is a key enzyme in the catabolism of the highly toxic compound oxalate, catalyzing the decarboxylation of oxalyl-CoA to formyl-CoA. The gene encoding a novel oxalyl-CoA decarboxylase from Bifidobacterium lactis DSM 10140 (oxc) was identified and characterized. This strain, isolated from yogurt, showed the highest oxalate-degrading activity in a preliminary screening with 12 strains belonging to Bifidobacterium, an anaerobic intestinal bacterial group largely used in probiotic products. The oxc gene was isolated by probing a B. lactis genomic library with a probe obtained by amplification of the oxalyl-CoA decarboxylase gene from Oxalobacter formigenes, an anaerobic bacterium of the human intestinal microflora. The oxc DNA sequence analysis revealed an open reading frame of 1,773 bp encoding a deduced 590-amino-acid protein with a molecular mass of about 63 kDa. Analysis of amino acid sequence showed a significant homology (47%) with oxalyl-CoA decarboxylase of O. formigenes and a typical thiamine pyrophosphate-binding site that has been reported for several decarboxylase enzymes. Primer extension experiments with oxc performed by using RNA isolated from B. lactis identified the transcriptional start site 28 bp upstream of the ATG start codon, immediately adjacent to a presumed promoter region. The protein overexpressed in Escherichia coli cross-reacted with an anti-O. formigenes oxalyl-CoA decarboxylase antibody. Enzymatic activity, when evaluated by capillary electrophoresis analysis, demonstrated that the consumption substrate oxalyl-CoA was regulated by a product inhibition of the enzyme. These findings suggest a potential role for Bifidobacterium in the intestinal degradation of oxalate.
Federici
Federica
F
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Vitali
Beatrice
B
Gotti
Roberto
R
Pasca
Maria Rosalia
MR
Gobbi
Silvia
S
Peck
Ammon B
AB
Brigidi
Patrizia
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002262:Carboxy-Lyases; C059418:oxalyl CoA decarboxylase