Enzymatic and mechanistic studies into tryptophan 2,3-dioxygenase

• Main Author: Thackray, Sarah J.
• Published: University of Edinburgh 2009
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662826

Tryptophan 2,3-dioxygenase (TDO) from <i>Xanthomonas campestris </i>(xcTDO) is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). TDO contains a histidine residue (histidine 55) in its active site, which hydrogen bonds to the indole nitrogen atom of L-tryptophan, and could function as an active site base. In this study we attempt to resolve the question of whether an active site base is necessary for catalytic activity, to which end, two complementary strategies, based on the active-site structure have been applied. Firstly, active-site mutants were studied, where histidine 55 was replaced by alanine or serine (H55A and H55S). The crystal structures of the H55A and H55S mutant forms were determined to 2.15 Å and 1.90 Å resolution respectively, in binary complexes with L-tryptophan. These structural data, in conjunction with potentiometric and kinetic studies, reveal that histidine 55 is not essential for turnover, but greatly disfavours the mechanistically unproductive binding of L-tryptophan to the oxidized enzyme, allowing control of catalysis. The second strategy utilises 1-methyl tryptophan. Our studies reveal that whilst 1-methyl tryptophan is not a substrate for TDO, and is not an inhibitor of its action, the active site mutants H55A and H55S can deoxygenate 1-methyl tryptophan. These catalytic differences are explained by comparison of the active sites of the enzymes, and support the proposal that a catalytic base is not necessary for enzymatic activity. In addition, these data provide new insight into the future of 1-methyl tryptophan as an inhibitor of IDO activity in cancer treatments.