Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

Erumbi S Rangarajan,Yunge Li,Eunice Ajamian,Pietro Iannuzzi,Stephanie D Kernaghan,Marie E Fraser,Miroslaw Cygler,Allan Matte

doi:10.1074/jbc.m510522200

Abstract

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

Highlights

Coenzyme A is a cofactor utilized by as many as 4% of all enzymes for a diverse variety of biological functions, including cell-cell-mediated recognition, nerve impulse conductance, transcription, and fatty acid biosynthesis and degradation [1, 2]
These studies established a landmark for the concept of substrate binding energy utilization by an enzyme to effect catalysis, showing that succinyl-CoA:3-oxoacid CoA-transferase (SCOT) utilizes its covalent (␥-glutamyl-CoA thioester) and noncovalent interactions with the CoA moiety of the acyl-CoA substrate differentially to reduce the Gibbs activation energy required for catalysis [13]
Crystal structures are available for three Family I CoA transferases, including glutaconate CoA transferase (GCT) from Acidaminococcus fermentens [14], acetate-CoA transferase (ACT, ␣-subunit) from Escherichia coli [15], and SCOT from pig heart [16, 17], no structure has yet been determined with bound substrate or product

Summary

Introduction

Coenzyme A is a cofactor utilized by as many as 4% of all enzymes for a diverse variety of biological functions, including cell-cell-mediated recognition, nerve impulse conductance, transcription, and fatty acid biosynthesis and degradation [1, 2]. I enzymes employ as acceptors 3-oxoacids, short-chain fatty acids, or glutaconate These enzymes operate with a ping-pong kinetic mechanism and form a covalent thioester intermediate [7]. A wealth of biochemical and mechanistic data are available for SCOT, largely based on the pioneering studies of Jencks and collaborators [7, 11,12,13] These studies established a landmark for the concept of substrate binding energy utilization by an enzyme to effect catalysis, showing that SCOT utilizes its covalent (␥-glutamyl-CoA thioester) and noncovalent interactions with the CoA moiety of the acyl-CoA substrate differentially to reduce the Gibbs activation energy required for catalysis [13].

Methods

Results

Conclusion