Novel l -Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101

Abstract

ABSTRACT 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.