Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones

Dao Feng Xiang, Peter Kolb, Alexander A. Fedorov, Chengfu Xu, Elena V. Fedorov, Tamari Narindoshivili, Howard J. Williams, Brian K. Shoichet, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)1762-1773
Number of pages12
JournalBiochemistry
Volume51
Issue number8
DOIs
StatePublished - Feb 28 2012

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Lactones
Sugars
Hydrolysis
Phosphates
Enzymes
Amidohydrolases
Substrates
4 alpha-glucanotransferase
Catalytic Domain
Listeria
Listeria monocytogenes
Bacilli
Bioinformatics
Cobalt
Computational Biology
Set theory
Bacillus
Libraries
Zinc
Hydrogen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Xiang, D. F., Kolb, P., Fedorov, A. A., Xu, C., Fedorov, E. V., Narindoshivili, T., ... Raushel, F. M. (2012). Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones. Biochemistry, 51(8), 1762-1773. https://doi.org/10.1021/bi201838b

Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones. / Xiang, Dao Feng; Kolb, Peter; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Narindoshivili, Tamari; Williams, Howard J.; Shoichet, Brian K.; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 51, No. 8, 28.02.2012, p. 1762-1773.

Research output: Contribution to journalArticle

Xiang, DF, Kolb, P, Fedorov, AA, Xu, C, Fedorov, EV, Narindoshivili, T, Williams, HJ, Shoichet, BK, Almo, SC & Raushel, FM 2012, 'Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones', Biochemistry, vol. 51, no. 8, pp. 1762-1773. https://doi.org/10.1021/bi201838b
Xiang DF, Kolb P, Fedorov AA, Xu C, Fedorov EV, Narindoshivili T et al. Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones. Biochemistry. 2012 Feb 28;51(8):1762-1773. https://doi.org/10.1021/bi201838b
Xiang, Dao Feng ; Kolb, Peter ; Fedorov, Alexander A. ; Xu, Chengfu ; Fedorov, Elena V. ; Narindoshivili, Tamari ; Williams, Howard J. ; Shoichet, Brian K. ; Almo, Steven C. ; Raushel, Frank M. / Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones. In: Biochemistry. 2012 ; Vol. 51, No. 8. pp. 1762-1773.
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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 {\AA} with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 {\AA} 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.",
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AU - Narindoshivili, Tamari

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AB - 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.

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