Crystal Structures of Amylosucrase from Neisseria polysaccharea in Complex with D-Glucose and the Active Site Mutant Glu328Gln in Complex with the Natural Substrate Sucro
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- 作者：Osman Mirza ; Lars K. Skov ; Magali Remaud-Simeon ; Gabrielle Potocki de Montalk ; Cecile Albenne ; Pierre Monsan ; and Michael Gajhede
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：July 31, 2001
- 年：2001
- 卷：40
- 期：30
- 页码：9032 - 9039
- 全文大小：203K
- 年卷期：v.40,no.30(July 31, 2001)
- ISSN：1520-4995
文摘
The structure of amylosucrase from Neisseria polysaccharea in complex with 
-D-glucosehas been determined by X-ray crystallography at a resolution of 1.66 Å. Additionally, the structure of theinactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0Å. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly inthe bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained4C1 conformation, and one that binds in the packing interface to a symmetry-related molecule. A thirdweaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientationof the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrosecomplex shows one molecule bound in the active site, where the glucosyl moiety is located at the 
-amylase-1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible accesschannel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtainedthrough enzyme interactions with the glucosyl ring and that an important part of the enzyme function isa precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328.The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, andArg509. Both Asp394 and Arg446 are located in an insert connecting -strand 7 and -helix 7 that ismuch longer in amylosucrase compared to other enzymes from the -amylase family (family 13 of theglycoside hydrolases).
-D-glucosehas been determined by X-ray crystallography at a resolution of 1.66 Å. Additionally, the structure of theinactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0Å. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly inthe bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained4C1 conformation, and one that binds in the packing interface to a symmetry-related molecule. A thirdweaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientationof the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrosecomplex shows one molecule bound in the active site, where the glucosyl moiety is located at the -amylase-1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible accesschannel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtainedthrough enzyme interactions with the glucosyl ring and that an important part of the enzyme function isa precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328.The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, andArg509. Both Asp394 and Arg446 are located in an insert connecting -strand 7 and -helix 7 that ismuch longer in amylosucrase compared to other enzymes from the -amylase family (family 13 of theglycoside hydrolases).