TY - JOUR

T1 - The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

AU - Champion, Elise

AU - Remaud-Simeon, Magali

AU - Skov, Lars Kobberøe

AU - Kastrup, Jette Sandholm

AU - Gajhede, Michael

AU - Mirza, Osman Asghar

N1 - Keywords: Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Glycoside Hydrolases; Models, Molecular; Protein Structure, Tertiary; Sucrose; Xanthomonas campestris

PY - 2009

Y1 - 2009

N2 - Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis-Menten kinetics, with a K(m) of 60.7 mM and a k(cat) of 21.7 s(-1). The structure of the enzyme was solved at a resolution of 1.9 A in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite -1 from the ;nonreducing' end. Binding of the glucosyl moiety in subsite -1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the ;nonreducing' end during the glucan-elongation cycle.

AB - Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis-Menten kinetics, with a K(m) of 60.7 mM and a k(cat) of 21.7 s(-1). The structure of the enzyme was solved at a resolution of 1.9 A in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite -1 from the ;nonreducing' end. Binding of the glucosyl moiety in subsite -1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the ;nonreducing' end during the glucan-elongation cycle.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1107/S0907444909040311

DO - 10.1107/S0907444909040311

M3 - Journal article

C2 - 19966417

VL - 65

SP - 1309

EP - 1314

JO - Acta Crystallographica Section D: Structural Biology

JF - Acta Crystallographica Section D: Structural Biology

SN - 2059-7983

IS - 12

ER -