Structural enzymology of oxalate degradation in Oxalobacter formigenes

Abstract

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.