Dataset Information

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.

SUBMITTER: Mazurkewich S

PROVIDER: S-EPMC4817193 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Publications

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 Scott S Brott Ashley S AS Kimber Matthew S MS Seah Stephen Y K SY

The Journal of biological chemistry 20160211 14

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 structural ...[more]

PMID: 26867578

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Project description:Background:Pseudomonas putida is a promising candidate for the industrial production of biofuels and biochemicals because of its high tolerance to toxic compounds and its ability to grow on a wide variety of substrates. Engineering this organism for improved performances and predicting metabolic responses upon genetic perturbations requires reliable descriptions of its metabolism in the form of stoichiometric and kinetic models. Results:In this work, we developed kinetic models of P. putida to predict the metabolic phenotypes and design metabolic engineering interventions for the production of biochemicals. The developed kinetic models contain 775 reactions and 245 metabolites. Furthermore, we introduce here a novel set of constraints within thermodynamics-based flux analysis that allow for considering concentrations of metabolites that exist in several compartments as separate entities. We started by a gap-filling and thermodynamic curation of iJN1411, the genome-scale model of P. putida KT2440. We then systematically reduced the curated iJN1411 model, and we created three core stoichiometric models of different complexity that describe the central carbon metabolism of P. putida. Using the medium complexity core model as a scaffold, we generated populations of large-scale kinetic models for two studies. In the first study, the developed kinetic models successfully captured the experimentally observed metabolic responses to several single-gene knockouts of a wild-type strain of P. putida KT2440 growing on glucose. In the second study, we used the developed models to propose metabolic engineering interventions for improved robustness of this organism to the stress condition of increased ATP demand. Conclusions:The study demonstrates the potential and predictive capabilities of the kinetic models that allow for rational design and optimization of recombinant P. putida strains for improved production of biofuels and biochemicals. The curated genome-scale model of P. putida together with the developed large-scale stoichiometric and kinetic models represents a significant resource for researchers in industry and academia.

Dataset Information

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440.

Publications

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440.

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