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Role of Trp140 at subsite –6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

¹ Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
² Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
³ National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

(RECEIVED September 29, 2005; FINAL REVISION November 18, 2005; ACCEPTED November 18, 2005)

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 4Å. 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.

Keywords: G6-amylase; crystal structure; site-directed mutagenesis; stacking interaction

Abbreviations: DP, degree of polymerization • EDTA, ethylenediaminetetraacetic acid • G1, glucose • G2, maltose • G3, maltotriose • G4, maltotetraose • G5, maltopentaose • G6, maltohexaose • G7, maltoheptaose • G8, malto-octaose • G9, maltononaose • HPLC, high-performance liquid chromatography

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051877006.

Reprint requests to: Kazuaki Harata, Biological Information Research Center, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; e-mail: k-harata{at}aist.go.jp; fax: +81-298-61-3444.

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