Thermophilic, reversible gamma-resorcylate decarboxylase from Rhizobium sp. strain MTP-10005: purification, molecular characterization, and expression.
We found the occurrence of thermophilic reversible gamma-resorcylate decarboxylase (gamma-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of gamma-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50 degrees C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k(cat)/K(m) (mMu(-1) . s(-1)) for gamma-resorcylate and resorcinol at 30 degrees C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the gamma-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and gamma-RDC probably catalyzes a reaction just before the hydroxylase in it does.
Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka-Fu 564-8680, Japan.
Masahiro
M
Yoshida
Nobuhiro
N
Fukuhara
Tadao
T
Oikawa
D000595:Amino Acid Sequence; D000818:Animals; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000032:analysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D062385:Hydroxybenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D051381:Rats; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004275:DNA, Ribosomal; D062385:Hydroxybenzoates; D012336:RNA, Ribosomal, 16S; D002262:Carboxy-Lyases; C412605:gamma-resorcylic acid