Comparative Analysis of Folding and Substrate Binding Sites between Regulated Hexameric Type II Citrate Synthases and Unregulated Dimeric Type I Enzymes^,

Abstract

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.

Probing the Roles of Key Residues in the Unique Regulatory NADH Binding Site of Type II Citrate Synthase of Escherichia coli

“…In this protein, residues 262-298 refold into a conformation that appears to be much of the way toward that required for a functional acetyl-CoA binding site. The evidence for this is a comparison with the structure of this region as it appears in the simpler, unregulated Type I CS enzymes (3,24). The structural element that provides the link between the R109L replacement and residues 262-298 appears to be the second polypeptide chain segment most perturbed in the R109L CS variant.…”

“…Type I and Type II CS subunits all have the same overall fold and very similar active sites, so that, as a first approximation, a Type II hexamer may be regarded as a trimer of Type I dimers (3). From this we have argued that NADH inhibition in the Type II enzymes is an evolutionary add-on; Type I dimers were altered by a series of mutations to generate NADH sites and new contact surfaces so that hexamers could form.…”

Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

“…Several metabolites were tested for their ability to inhibit or stimulate citrate synthase activity. NADH strongly inhibits prokaryotic hexameric citrate synthases (35,43) but had no effect on the activity of the Geobacter citrate synthase at physiologically relevant concentrations (1 to 5 mM). 2-Oxoglutarate, also a specific inhibitor of some hexameric citrate synthases (56), did not inhibit the Geobacter enzyme.…”

A Specialized Citric Acid Cycle Requiring Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase (AarC) Confers Acetic Acid Resistance on the Acidophile Acetobacter aceti

“…Citrate synthase (40) and Mdh (57) are susceptible to allosteric and product inhibition, respectively. However, both the acid-tolerant H. pylori and the acidophilic A. aceti contain citrate synthase forms that are insensitive to NADH (15,44).…”