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Functional annotation and structural characterization of a novel lactonase hydrolyzing D-xylono-1,4-lactone-5-phosphate and L-arabino-1,4-lactone-5-phosphate

Korczynska M, Xiang DF, Zhang Z, Xu C, Narindoshvili T, Kamat SS, Williams HJ, Chang SS, Kolb P, Hillerich B, Sauder JM, Burley SK, Almo SC, Swaminathan S, Shoichet BK, Raushel FM (2014) Biochemistry 53, 4727-4738. PMCID: PMC4108184

This study from the AH Bridging Project, Computation Core, and Structure Core exemplifies the necessity of integrating orthogonal techniques (genome context, docking, enzyme structure, kinetic analysis) in order to predict substrates for previously uncharacterized members of the Amidohydrolase Superfamily. This enzyme superfamily is particularly challenging as it exhibits very high substrate specificity where alterations at a single stereocenter distinguish the difference between active and non-active substrates.


A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (2) with kcat/Km values of 4.7 × 10(4) and 5.7 × 10(4) M(-1) s(-1) and l-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 × 10(4) and 2.2 × 10(4) M(-1) s(-1), respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members. 

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2014 KorczynskaAbstract Image.

2014 KorczynskaFigure 1: Sequence similarity network organization showing one of the 24 clusters representing the amidohydrolase superfamily sequences annotated as belonging to Cog1735.

2014 KorczynskaScheme 1

2014 Korczynska Scheme 2

2014 KorczynskaFigure 2: Ribbon representation of the three-dimensional structure of the (beta/alpha)8-barrel fold of a single chain of Ms0025 (PDB entry 3MSR.3OVG). The two Zn2+ metals are colored gray, and the phosphate ligand is colored orange. The central beta-barrel is colored purple, and the helices are colored cyan.

2014 KorczynskaFigure 3:Binuclear metal center of Ms0025 (PDB entry 3OVG). Structural model for the active site showing residues that coordinate the binuclear metal center. All contacts between the two divalent zinc ions (gray spheres), water (red spheres), or phosphate (orange sticks) and active site residues (white sticks) are indicated with dashed lines. The alpha-zinc is coordinated by His24, His26, Kcx153, Asp272, and two water molecules, while the beta-zinc exhibits tetrahedral geometry and is coordinated by Kcx153, His186, His214, and the bridging water molecule.

2014 KorczynskaFigure 4:Gene organization of the operon and ulaA-G regulon involved in L-ascorbate metabolism in E. coli, which encodes a phosphotransferase system (PTS) (ulaABC genes).

2014 KorczynskaFigure 5:Comparison of lactonase structures of Ms0025 (PDB entry 3OVG) and UlaG (PDB entry 3BV6/2WYL).

2014 KorczynskaFigure 6:Docked compounds.

Reprinted with permission from Korczynska et al. Biochemistry 53, 4727-4738. Copyright 2014 American Chemical Society.