title: 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 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.
authors: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Scott
authors: S
authors: Mazurkewich
authors: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Ashley S
authors: AS
authors: Brott
authors: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Matthew S
authors: MS
authors: Kimber
authors: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada sseah@uoguelph.ca.
authors: Stephen Y K
authors: SY
authors: Seah
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D062385:Hydroxybenzoates; C009091:protocatechuic acid
keywords: LigJ; PIG-L deacetylase; Pseudomonas; bacterial metabolism; enzyme mechanism; gallate; hydratase; lignin degradation; metalloenzyme; protocatechuate