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 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-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.

Rapid, Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans

“…The conversion of 3MGA into 4-oxalomesaconate (OMA) requires two steps, namely, ringopening and demethylation. In the first pathway, LigM demethylates 3MGA to gallate, followed by oxidation by DesB or LigAB to yield OMA (27)(28)(29). In the second pathway, DesZ or LigAB oxidizes and demethylates 3MGA to 2-pyrone-4,6-dicarboxylate (PDC), followed by ring hydrolysis using LigI (30).…”

Isolation, identification and characterization of lignin‐degrading bacteria from Qinling, China

“…In particular, P. putida KT2440 strain, similar to our selections of Y2 and Y7, was shown to catabolize lignin and the cleavage pathways have been studied (Mazurkewich et al . ). Ochrobactrum and Rhizobium isolates have already been isolated from woodland soil incubated in enrichment cultures containing wheat straw lignocellulose.…”

Investigation into the Mode of Phosphate Activation in the 4-Hydroxy-4-Methyl-2-Oxoglutarate/4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase from Pseudomonas putida F1

“…The intracellular concentration of P i in the Pseudomonads and Sphingomonads which contain these pathways is likely to be in the low millimolar range and would contribute to the aldolases normal physiological functioning [ 40 ]. In the gallate degradation pathway of Pseudomonas putida , the turnover rate of the enzymes are comparable to that of the native HMG/CHA aldolase (GalA = 42 s -1 , GalD = unknown, and GalB = 2 to 14 s -1 depending on the metal utilized) [ 41 , 42 ]. Changes in cellular P i content would likely not affect gallate utilization.…”