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The complex crystal structure of Saccharomyces cerevisiae dihydrolactase and inhibitor 5-fluorolactate reveals a new binding mode

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Document pages: 9 pages

Abstract: Dihydrolactase (dhoase) has a binuclear metal center in which two zinc ions are bridged by post-translational aminoacylated lysine. Dhoas catalyzes the reversible cyclization of N-carbamoyl aspartic acid (CA ASP) to dihydrowhey acid (DHO) in the third step of pyrimidine nucleotide biosynthesis pathway, which is an attractive target for potential anticancer and antimalarial chemotherapy. The crystal structure of ligand bound DHO enzyme shows that when CA ASP binds (loop in mode) or leaves the active site, the flexible ring extends to the active site and promotes the release of product DHO (loop out mode). Dhoase binds products similar to the inhibitor 5-fluoroborate (5-foa) in a similar manner to DHO. In this study, we reported the crystal structure of dhoas extracted from molasseses cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

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