Public Abstract

DE-SC0018143: Novel Microbial Based Enzymatic CO2 Fixation Mechanisms: Conformational Control of Enzymatic Reactivity

Award Status: Expired

Institution: Washington State University, Pullman, WA
UEI: XRJSGX384TD6
DUNS: 041485301

Most Recent Award Date: 07/26/2022
Number of Support Periods: 5
PM: McLean, Barbara Gail

Current Budget Period: 09/01/2021 - 08/31/2023
Current Project Period: 09/01/2019 - 08/31/2023
PI: Peters, John

Supplement Budget Period: N/A

Public Abstract

Novel microbial based enzymatic CO2 fixation/carboxylation mechanisms: Conformational control of enzymatic reactivity John W. Peters, Principal Investigator Institute of Biological Chemistry, Washington State University, Clark Hall, Pullman, WA 99164 Jennifer L. DuBois, Co-Principal Investigator Montana State University, Chemistry and Biochemistry Bldg., Bozeman, MT 59717 Brian Bothner, Co-Principal Investigator Montana State University, Chemistry and Biochemistry Bldg., Bozeman, MT 59717 The broad primary long term goal of the proposed research is to provide insight into the mechanism of novel carboxylation reactions catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. Aliphatic alkenes as well as aromatic compounds are substrates for microsomal cytochrome P450 oxygenases. These compounds are oxygenated across double bonds to the corresponding epoxides. The epoxides formed serve as alkylating reagents that can covalently modify both proteins and nucleic acids and result in mutagenic effects. A novel bacterial pathway of the aerobic microorganism Xanthobacter autotrophicus strain Py2 has recently been described in which epoxides produced by alkene oxidation and ketones produced by the oxidation of alcohols are further metabolized to beta-keto acids. The discovery of the pathway was facilitated by the ability of the organism to use aliphatic alkenes, epoxides, and ketones as sole sources of carbon and energy in spite of the toxicity of these compounds. Studies of alkene and ketone metabolism have identified a central role for CO₂ and, specifically, CO₂ fixation reactions in these processes. The CO₂ fixing enzymes of the pathways are distinct carboxylases with unique molecular properties and cofactor requirements. The main focus and aims of this proposal involve providing the structural basis for the mechanistic understanding of the reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase / carboxylase and acetone carboxylase, the terminal steps in the convergent pathways of alkene and ketone metabolism in Xanthobacter autotrophicus strain Py2. We are utilizing a multidisciplinary approach involving integrating kinetic studies, site specific amino acid substitution studies, and structural work involving x-ray crystallography and mass spectrometry in the presence of substrate analogs and mechanism base inhibitors to ascertain information concerning the biochemical mechanisms of enzyme catalyzed reactions.

Novel microbial based enzymatic CO2 fixation/carboxylation mechanisms: Conformational control of enzymatic reactivity

John W. Peters, Principal Investigator

Institute of Biological Chemistry, Washington State University, Clark Hall, Pullman, WA 99164

Jennifer L. DuBois, Co-Principal Investigator

Montana State University, Chemistry and Biochemistry Bldg., Bozeman, MT 59717

Brian Bothner, Co-Principal Investigator

Montana State University, Chemistry and Biochemistry Bldg., Bozeman, MT 59717

The broad primary long term goal of the proposed research is to provide insight into the mechanism of novel carboxylation reactions catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. Aliphatic alkenes as well as aromatic compounds are substrates for microsomal cytochrome P450 oxygenases. These compounds are oxygenated across double bonds to the corresponding epoxides. The epoxides formed serve as alkylating reagents that can covalently modify both proteins and nucleic acids and result in mutagenic effects. A novel bacterial pathway of the aerobic microorganism Xanthobacter autotrophicus strain Py2 has recently been described in which epoxides produced by alkene oxidation and ketones produced by the oxidation of alcohols are further metabolized to beta-keto acids. The discovery of the pathway was facilitated by the ability of the organism to use aliphatic alkenes, epoxides, and ketones as sole sources of carbon and energy in spite of the toxicity of these compounds. Studies of alkene and ketone metabolism have identified a central role for CO₂ and, specifically, CO₂ fixation reactions in these processes. The CO₂ fixing enzymes of the pathways are distinct carboxylases with unique molecular properties and cofactor requirements. The main focus and aims of this proposal involve providing the structural basis for the mechanistic understanding of the reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase / carboxylase and acetone carboxylase, the terminal steps in the convergent pathways of alkene and ketone metabolism in Xanthobacter autotrophicus strain Py2. We are utilizing a multidisciplinary approach involving integrating kinetic studies, site specific amino acid substitution studies, and structural work involving x-ray crystallography and mass spectrometry in the presence of substrate analogs and mechanism base inhibitors to ascertain information concerning the biochemical mechanisms of enzyme catalyzed reactions.