5cgz

Publication Abstract from PubMed

Bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of ligno-biomass. The gallate degradation pathway of Pseudomonas putida KT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB) which has 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetyl glucosamine deacetylase family, and is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereo-specificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contained bound Zn2+ cofactor; however the enzyme is capable using Fe2+ and Co2+ with similar efficiencies. 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 x 104 fold and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependency studies of the wild-type and variant enzymes suggests roles for the Glu48 and His164 in the catalytic mechanism. Comparison to the PIG-L deacetylases has led to a proposed mechanism for GalB wherein Glu48 positions and activates the metal ligated water for the hydration reaction and His164 acts as a catalytic acid.

Structural and Kinetic Characterization of the 4-Carboxy-2-Hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440.,Mazurkewich S, Brott AS, Kimber MS, Seah SY J Biol Chem. 2016 Feb 11. pii: jbc.M115.682054. PMID:26867578^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.