Role of Trp140 at Subsite -6 on the Maltohexaose Production of Maltohexaose-producing Amylase from Alkalophilic Bacillus Sp.707.
From: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Protein science : a publication of the Protein Society
- Publish Date: Mar 2006
- ISSN: 0961-8368
- Volume: 15
- Issue: 3
- Pages: 468-77
- Medium: Print
- Language: English
- Citation (JAMA): Kanai Ryuta, Haga Keiko, Akiba Toshihiko, et al. Role of Trp140 at Subsite -6 on the Maltohexaose Production of Maltohexaose-producing Amylase from Alkalophilic Bacillus Sp.707.. Protein Sci. Mar 2006;15:468-77
Abstract
Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP=17). Our previous crystallographic study showed that G6-amylase has nine subsites, from -6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites -6 to -2, while G1 and G6 are found at subsites +2 and -7 to -2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite -6 is stacked to the indole moiety of Trp140 within a distance of 4A. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite -6 and to govern product specificity for G6 production.
Mesh Headings (Keywords): Bacillus, Bacterial Proteins, Binding Sites, Catalytic Domain, Hordeum, Ligands, Models, Molecular, Mutagenesis, Site-Directed, Oligosaccharides, Tryptophan, alpha-Glucosidases
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