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

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 M, 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.

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para‐hydroxybenzoate catabolism

“…The findings were also consistent with several previous reports of MHB 6‐hydroxylase‐encoding genes clustered with genes for GA degradation (Liu et al ., ; Liu and Zhou, ). Indeed, PHB‐7a was found to also grow on MHB as the sole source of carbon and energy.…”

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58

“…The protein concentration was determined by the Bradford method with bovine serum albumin as the standard. The native molecular weights of expressed proteins were determined as described previously (54). Briefly, the molecular weight of PcaA1A2 was measured using fast protein LC and detected at 280 nm.…”

A Two-Component para -Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

“…pnpA1 and pnpA2 amplified by PCR with primers in Table 2 from genomic DNA of strain RKJ300 were cloned into pET-28a to obtain the expression constructs listed in Table 1. The plasmids were then transformed into E. coli Rosetta(DE3)/pLysS for protein expression and purification as described previously (21).…”