The essential metabolic enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GAP). DXP is an essential branch point metabolite in bacteria, as it is a precursor to isoprenoids, as well as the important cofactors thiamine diphosphate (ThDP) and pyridoxal phosphate. DXP synthase (DXPS) is not present in humans, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that could be exploited for selective inhibition of DXP synthase over mammalian ThDP-dependent enzymes. The large active site and unique domain arrangement of DXP synthase in addition to its novel random sequential mechanism are particular features of DXP synthase that can guide selective targeting strategies. In addition, we have shown that DXPS is promiscuous in terms of its acceptor substrate, as it can utilize several aldehydes and nitroso analogues, but previous reports have suggested a strict requirement for pyruvate as the donor substrate in the presence of the natural acceptor substrate, D-GAP. In this study, we show that DXPS can indeed utilize alternative donor substrates, and propose that donor substrate usage may be dictated by the nature and availability of acceptor substrate. We demonstrate that although hydroxypyruvic acid is not efficiently coupled with d-GAP as an acceptor, it is turned over in the presence of O<sub>2</sub> to produce α-hydroxyperacetate. This alternative substrate pair on DXP synthase sets a precedent for unique donor/acceptor combinations which could have important implications for DXP synthase function in bacterial cells. These results highlight a unique toolset for studies of donor substrate specificity and advance our knowledge of alternative chemistries of DXP synthase which are valuable to guide inhibitor design and understand function.

Understanding the alternative activities of DXP synthase

254th National Meeting (2017)

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