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

Abstract

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). Mainly, these reactions involve the binding and transfer of an acyl group from one substrate to another as part of an enzymatic reaction; it has been noted that coenzyme A is the most prominent acyl group carrier in all living systems (3). Enzymecatalyzed reactions employing CoA thioesters can be divided into two categories, (i) those where the thioester carbonyl C atom reacts as an electrophile and (ii) those where the thioester ␣-carbon is deprotonated and reacts as a nucleophile, in Claisen enzymes (1). CoA transferases, which catalyze the reversible transfer of CoA from a donor CoA thioester to a carboxylic acid acceptor generating the free donor and a new acyl-CoA (Scheme 1), belong to the first category of enzymes. Among the large number of CoA transferases, much attention has focused on mitochondrial succinyl-CoA:3-oxoacid CoA-transferase (SCOT), 4 as its autosomal recessive deficiency in humans results in improper ketone body utilization causing episodic severe ketosis, hypoglycemia, and ultimately coma (4, 5).Three classes of CoA transferases have been defined based mainly on mechanistic and sequence criteria (6). Family 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). The most thoroughly studied member of the Family I CoA transferases is SCOT. Family II consists of the multifunctional enzymes citrate or citramalate lyase, and unlike Family I enzymes, they do not form a covalent thioester intermediate. Family III enzymes have been discovered more recently, and are distinct both mechanistically (6, 8) and structurally (9) 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). The utilization of this binding energy for catalysis differs for different chemical moieties within the CoA cofactor, as well for the different steps along the reaction coordinate. Although 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. The absence of an enzyme-substrate co-crystal structure for any Family I CoA transferase has prevented...

Reinvestigation of the Catalytic Mechanism of Formyl-CoA Transferase, a Class III CoA-transferase

“…4B). This is the first confirmation of the existence of the ␤-aspartyl-CoA thioester intermediate in catalysis for a Class III CoA-transferase, but similar mass spectroscopic analyses have been performed for several Class I enzymes (13,14,30).…”

“…The formation of covalent intermediates involving a glutamate residue of the enzyme results in a classical ping-pong mechanism with exchanging substrate/ product glutamyl-acyl anhydrides and ␥-glutamyl-CoA thioesters (12,13). The ␥-glutamyl-CoA thioester was first identified in a Class I transferase in 1968 by electrophoresis and chromatography studies with labeled borohydride (12) and was recently trapped in a crystallographic study, giving the first structural proof of its existence (14).…”

Crystal structure of the complex between 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and CoA

“…The acyl-CoAT from E. coli also holds a glutamyl-CoA-thioester (Rangarajan et al 2005). The binding modes of both CoA-thioesters have been compared (Macieira et al 2009).…”

“…The crystal structures of 4-HB-CoATs from Shewanella oneidensis (PDB-code: 2OAS) and Porphyromonas gingivalis (PDB-code: 3D3U) have been solved prior to the clostridial structure by the North East Structural Genomics consortium (http://www.nesg.org) but not published yet. A secondary structure matches search with the MSDfold (Krissinel and Henrick 2004) of 4-HB-CoAT from C. aminobutyricum as query structure in the protein data bank (http://www.rcsb.org/) revealed 6 further known CoAT structures, namely a putative CoAT from Pseudomonas aeruginosa (PDB-code: 2G39), a putative acetylCoA hydrolase/transferase PG1013 from P. gingivalis (PDB-code: 2NVV), acyl-CoAT from E. coli (YDIF) (PDB-code: 2AHV) (Rangarajan et al 2005), succinylCoA: 3-oxoacid CoA-transferase from pig heart (SCOT) (Bateman et al 2002), 3-oxoacid CoAT-1 from Homo sapiens (PDB-code: 3DLX) and glutaconate CoAT from A. fermentas (Jacob et al 1997) (all structures with no reference have been deposited with the protein data bank). The structure of the putative a-subunit of citrate lyase from Streptococcus mutans (PDB-code: 2HJ0) is also related, which led us to reclassify the CoA-transferases (Macieira et al 2009).…”

Proteomic survey towards the tissue-specific proteins of mouse mitochondria

“…Hence, this observation questions how the mitochondrial trifunctional protein functions in kidney mitochondria, or the function of HADHB is not fully understood. SCOT is a mitochondrial matrix homodimer that was first identified in pig hearts and sheep kidneys [30,31]. SCOT catalyzes the reversible transfer of a CoA moiety from succinyl CoA to the ketone body acetoacetate [32].…”