Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch |
| |
Authors: | Tan Tien-Chye Mijts Benjamin N Swaminathan Kunchithapadam Patel Bharat K C Divne Christina |
| |
Affiliation: | 1 KTH School of Biotechnology, AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden 2 Microbial Gene Research and Resource Facility, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Queensland 4111, Australia 3 Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore |
| |
Abstract: | The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a 2So skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch. |
| |
Keywords: | N domain, N-terminal domain α-Amylase, α-1,4-glucan 4-glucanohydrolase GH, glycoside hydrolase PDB, Protein Data Bank CBM, carbohydrate-binding module VAE, valienamine 6DG, 6-deoxy- smallcaps" >d-glucoside DP, degree of polymerization Dns-Cl, dansyl chloride Mes, 4-morpholineethanesulfonic acid PEG, polyethylene glycol TLS, translation, libration, screw rotation |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|