Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones (original) (raw)

ArticleFebruary 8, 2012

Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones

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Biochemistry

Cite this: Biochemistry 2012, 51, 8

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Published February 8, 2012

research-article

Copyright © 2012 American Chemical Society

Abstract

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Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, d-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The _k_cat/_K_m values for the cobalt-substituted enzymes with these substrates are ∼105 M–1 s–1.

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Copyright © 2012 American Chemical Society

Supporting Information

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A figure showing the enrichment of the most frequently observed reaction types from the docking of the HEI KEGG database to the structure of Lmo2620 (Figure S1), a list of additional lactones tried as potential substrates (Table S1), and a list of proteins expected to have the same substrate profile as Lmo2620 and Bh0225 (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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Biochemistry

Cite this: Biochemistry 2012, 51, 8

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Published February 8, 2012

Copyright © 2012 American Chemical Society

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