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. 2012 Feb 28;51(8):1762-73.
doi: 10.1021/bi201838b. Epub 2012 Feb 15.

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

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Free PMC article

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

Dao Feng Xiang et al. Biochemistry. .
Free PMC article

Abstract

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 ~10(5) M(-1) s(-1).

Figures

Figure 1
Figure 1
Cytoscape network representation (www.cytoscape.org) of the sequence relationships in cog1735 from the amidohydrolase superfamily. Each node in the network represents a single sequence and each edge (depicted as lines) represents the pairwise connection between two sequences with the most significant BLAST E-value (better then 1x10−80). Lengths of edges are not meaningful except that sequences in tightly clustered groups are relatively more similar to each other than sequences with few connections. The triangular nodes represent proteins that have been functionally and/or structurally characterized. Additional information is provided in the text.
Figure 2
Figure 2
Ribbon representation for the three-dimensional structure of [Zn/Zn]-Lmo2620 with two phosphates in the active site.
Figure 3
Figure 3
Active site of [Zn/Zn]-Lmo2620 with the two bound phosphates. The two divalent zinc ions are presented as green spheres.
Figure 4
Figure 4
Time dependent hydrolysis of d-lyxonolactone-5-phosphate catalyzed by Bh0225 followed by 31P-NMR spectroscopy at pH 7.1 and 5 °C. (A), 0 minutes; (B), 5 minutes; (C), 20 minutes; (D), 35 minutes; (E), 60 minutes.
Figure 5
Figure 5
Docked poses of characteristic high-ranking candidate substrates from the docking screen of the high-energy intermediate form of the KEGG library. (A) A sugar lactone, compound K5 in Table 3, ranked 217 in the docking screen. (B) A lactone carboxylate, ranked 91 in the docking screen. The high energy intermediate form of both compounds was docked and is represented; this group interacts with the catalytic metals. Key recognition residues are labeled. Putative hydrogen bonds are represented as amber dashed lines. Protein carbons are gray, ligand carbons green, oxygens are red nitrogens are blue.
Figure 6
Figure 6
Docked poses of the substrates with Lmo2620. The position of the phosphate ion from the original structure is shown in cyan; the protein and ligands are otherwise colored as in Figure 5. (A) Compound 1. (B) Compound 8.
Figure 7
Figure 7
Amino acid sequence alignment of selected proteins from cog1735. Lmo2620 (group 5), Bh0225 (group 5), Dr0930 (group 7), phosphotriesterase homology protein (group 1) from E. coli (locus tag: b3379) and MS53_0025 from Mycoplasma synoviae 53 (group 6). The metal binding ligands are shown in red. The residues that have been shown to facilitate the binding of substrates in the active site of Lmo2620 and Bh0225 are highlighted in green. The corresponding residues in the other three proteins are highlighted in gray.
Scheme 1
Scheme 1
Scheme 2
Scheme 2

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