Structural enzymology of oxalate degradation in Oxalobacter formigenes

Oxalic acid, as one of nature's most highly oxidised compounds, is toxic to most organisms. It is introduced in the human body in the diet but also as a waste product of cellular metabolism. Mammals do not posses the ability to degrade oxalate and must excrete it in the urine or through the intestine. Accumulation of oxalate may lead to a number of pathological conditions in humans and a majority of all kidney stones are formed by calcium oxalate. Fortunately, the anaerobic bacterium Oxalobacter formigenes has been shown to play a key role in the mammalian oxalate homeostasis. The bacterium, which inhabits the gastrointestinal tract of most vertebrates including humans, has evolved a method for oxalate catabolism and degrades it in a two enzyme pathway releasing formate and carbon dioxide.

This thesis presents structural characterisation of the two enzymes active in oxalate catabolism in O. formigenes, oxalyl-CoA decarboxylase (OXC) and formyl-CoA transferase (FRC). FRC catalyses the activation of oxalate in the form of oxalyl- CoA by transferring a CoA carrier from formyl-CoA. OXC, the second enzyme of the pathway, decarboxylates oxalyl-CoA releasing carbon dioxide and regenerating formyl-CoA.

The three-dimensional structure of OXC was determined to 1.73 Å resolution from a merohedrally twinned crystal. As a thiamin diphosphate-dependent enzyme, OXC displays the conserved fold consisting of three alpha/beta-domains with the coenzyme bound in a strictly conserved conformation between two subunits. A novel set of active site residues was observed for OXC, and the identification of an ADP molecule bound in the regulatory domain of the protein led to the discovery that ADP is an efficient activator of OXC. Several structures of OXC complexes have been determined, including a substrate complex with an inactive coenzyme analogue, a product complex and a reaction intermediate obtained by freezetrapping experiments. A catalytic mechanism is presented based on a combination of structural features and mutagenesis data.

FRC, as a Class III CoA-transferase, is a homodimeric enzyme with a peculiar fold consisting of two monomers interlocking each other like links of a chain. By freezetrapping crystallography we have identified a previously undiscovered intermediate in the catalytic reaction of FRC, leading to reinterpretation of the catalytic mechanism. Active site features in structures of several reaction intermediates and point-mutated variants are combined to present a plausible scenario for the catalytic steps. Finally, we demonstrate that a protein annotated as a putative formyl-CoA transferase in Escherichia coli is indeed a FRC ortholog, and the substrate specificity and kinetic behaviour of the two enzymes are compared.

List of scientific papers

I. Berthold CL, Sidhu H, Ricagno S, Richards NG, Lindqvist Y (2006). "Detection and characterization of merohedral twinning in crystals of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes." Biochim Biophys Acta 1764(1): 122-8. Epub 2005 Sep 12
https://pubmed.ncbi.nlm.nih.gov/16198641

II. Berthold CL, Moussatche P, Richards NG, Lindqvist Y (2005). "Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate." J Biol Chem 280(50): 41645-54. Epub 2005 Oct 10.
https://pubmed.ncbi.nlm.nih.gov/16216870

III. Berthold CL, Toyota CG, Moussatche P, Wood MD, Leeper F, Richards NG, Lindqvist Y (2007). "Crystallographic snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases." Structure 15(7): 853-61
https://pubmed.ncbi.nlm.nih.gov/17637344

IV. Berthold CL, Toyota CG, Richards NGJ, Lindqvist Y (2007). "Re-investigation of the catalytic mechanism of formyl-CoA transferase, a Class III CoAtransferase." Journal of Biological Chemistry (Submitted)

V. Toyota CG, Berthold CL, Gruez A, Jónsson S, Lindqvist Y, Cambillau C, Richards NGJ (2007). "Differential substrate specificity and kinetic behavior in Escherichia coli YfdW and Oxalobacter formigenes Formyl-CoA transferase." Journal of Bacteriology (Submitted)