Insights into the function of RifI2: Structural and biochemical investigation of a new shikimate dehydrogenase family protein |
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Authors: | James Peek Christel Garcia John Lee Dinesh Christendat |
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Affiliation: | 1. Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2;2. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada M5G 1 L7 |
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Abstract: | The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD+ instead of NADP+ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD+ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD+-dependent member of the SDH family. |
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Keywords: | SDH, shikimate dehydrogenase SdhL, shikimate dehydrogenase-like Ael1, AroE-like1 AHBA, 3-amino-5-hydroxybenzoic acid IPTG, isopropyl ß -d-thiogalactopyranoside Ni-NTA, nickel-nitriloacetic acid R.M.S.D., root mean square deviation |
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