Novel L-cysteine-dependent maleylpyruvate isomerase in the gentisate pathway of Paenibacillus sp. strain NyZ101.
Glutathione- and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G+C Gram-positive strains. In the present study, a low-G+C Gram-positive Paenibacillus sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (bagKLIX) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with L-cysteine, cysteinylglycine, or glutathione, as its cofactor. The K(m) values of these three thiols for BagL were 15.5, 8.4, and 552 μM, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G+C Gram-positive strains, BagL should utilize L-cysteine as its physiological cofactor in vivo. The addition of Ni(2+) increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni(2+) ion and were necessary for its enzyme activity. BagL is the first characterized L-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Ting-Ting
TT
Liu
Ning-Yi
NY
Zhou
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D023061:Gene Order; D005841:Gentisates / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019745:cis-trans-Isomerases / Q000378:metabolism*
D004269:DNA, Bacterial; D005841:Gentisates; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C032948:3-hydroxybenzoic acid; D019745:cis-trans-Isomerases; C046138:maleylpyruvate isomerase; D003545:Cysteine