Abstract
The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of ligno-biomass. The gallate degradation pathway ofPseudomonas putidaKT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) fromSphingomonassp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-LN-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn(2+)cofactor; however the enzyme is capable of using Fe(2+)and Co(2+)with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 × 10(4)-fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid.
Highlights
Lignin, a complex biopolymer found in the plant cell wall, represents a large carbon reservoir in the environment
Overall Structure of GalB—An initial substructure of GalBNt was determined by zinc single-wavelength anomalous diffraction (SAD) phasing to 2.2 Å resolution, which was subsequently utilized as a model for molecular replacement of the native data set (2.1 Å) (Table 1)
The metabolism of gallic acid by P. putida through the gallate pathway is similar to the metabolism of other aromatic compounds such as hydroxyphenyl acetate and catechol
Summary
Ase (TIM) barrel that is structurally distinct from other characterized divalent metal-dependent hydratases [13,14,15]. Like LigJ, GalB has no sequence similarity to the previously characterized divalent metal-dependent hydratases. No structural or mechanistic investigations have been reported in the literature to elucidate the mechanism of GalB and its relationship to LigJ CHM hydratases. We report the first structure and in-depth kinetic characterization of the GalB CHM hydratase from Pseudomonas putida KT2440. We show that both GalB and LigJCsE6 catalyze the reversible hydration of 4-carboxy-2-hydroxypenta-2,4-dienoate (CHPD; a CHM analog lacking the C5 carboxylate) to HMG and propose a previously undescribed route for HMG production from the metabolism of the lignin metabolite 5,5Ј-dehydrodivanillate (DDVA). Our findings shed light on the analogous enzymes GalB and LigJ, which have evolved convergently to perform the same chemical reaction
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