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Role of Trp140 at subsite −6 on the maltohexaose production of maltohexaose‐producing amylase from alkalophilic Bacillus sp.707
Author(s) -
Kanai Ryuta,
Haga Keiko,
Akiba Toshihiko,
Yamane Kunio,
Harata Kazuaki
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051877006
Subject(s) - amylase , chemistry , residue (chemistry) , moiety , stereochemistry , indole test , enzyme , biochemistry
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.