Structural and kinetic characterization of the 4-carboxy-2-hydroxymuconate hydratase from the gallate and protocatechuate 4, 5-cleavage pathways of Pseudomonas putida KT2440

Abstract

The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of lignobiomass. The gallate degradation pathway of Pseudomonas putida KT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identifiedCHMhydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-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 acommonmetabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn2+ cofactor; however the enzyme is capable of using Fe2+ and Co2+ 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 × 104-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.