Determination And Analysis Of Crystal Structure Of Urate Oxidase From Bacillus Subtilis 168

Urate oxidase is an oxygenase that has long been proposed to act in the presence of copper ions to catalyze the degradation of uric acid to allantoin.The enzyme was later believed to act in a cofactor independent manner,when N'colloc'h and colleagues solved the first ever crystal structure of urate oxidase-inhibitor complex from Aspergillus flavus at 2.05A resolution.Urate oxidase is absent in humans due to the genetic evolution over time.Urate oxidase works by catalyzing the oxidative degradation of uric acid to allantoin via peroxide formation by a radical recombination mechanism.The enzyme is gaining much more importance in the medical and research field due to its beneficial effects against Gout.The disease is the painful swelling of joints and joint cavities that affects mostly women in their adult age in almost every population.Abnormally increased levels of uric acid are the hallmark of gout which occurs due to the genetic errors in the enzymes and transporters regulating the uric acid homeostasis in the body.The abnormal increase in uric acid can be treated by injecting urate oxidase into the human body.Researchers have suggested urate oxidase to be a better medicine for the cure of Hyperuricemia and Gout.Fasturtec is the market name of the AfUOX heterologously expressed in Saccharomyces cerevisiae but it is associated with some serious side effects of methemoglobinemia in Caucasian patients.The homotetrameric structure of urate oxidase have been reported previously from some bacterial species proposing that the homotetramer is essential for enzyme activity and stability.The active site residues from the substrate binding pocket had been studied and are still under investigation to unravel the mechanism of urate oxidase activity at atomic level.Here we have solved the crystal structure of urate oxidase(4-310 residues)from Bacillus subtilis 168 at 2.6A resolution.We performed Comparative analysis of BsUOX by structure alignment with complex crystal structure of urate oxidase-uric acid from Aspergillus flavus(PDB entry 4D12)which showed some conserved BsUOX amino acid residues Thr69,Ser243,Gln245,and Asn271 in the active site region that can potentially bind uric acid.We predicted some residues such as Ile244 and Gln299 that may interact with the uric acid via hydrophobic interactions which needs further confirmation.Our structure has an unusual C-terminal fold comprising of four residues(Arg305,Glu306,Asp307 and Ala308)that may be involved in BsUOX thermo stabilization.Our SAXS data revealed that the BsUOX adopts a homotetramer conformation in both crystal and solution forms.The residues at the tetrameric interface may also contribute towards BsUOX catalytic activity and stability.In addition,the side chain replacement of wild type BsUOX(in pyMOL)proposed by Li et al.,2017 to mutated BsUOX residues(D44V,Q268R,K285Q and H300R)suggested that these residues may not affect the enzyme's catalytic activity due to their distant locations.This work will be helpful for further functional and biochemical studies of the enzyme for future drug design and development against Gout and Hyperuricemia.