Biochemical and crystallographic analyses of maltohexaose-producing amylase from alkalophilic Bacillus sp. 707.
Maltohexaose-producing amylase, called G6-amylase (EC 3.2.1.98), from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) from starch and related alpha-1,4-glucans. To elucidate the reaction mechanism of G6-amylase, the enzyme activities were evaluated and crystal structures were determined for the native enzyme and its complex with pseudo-maltononaose at 2.1 and 1.9 A resolutions, respectively. The optimal condition for starch-degrading reaction activity was found at 45 degrees C and pH 8.8, and the enzyme produced G6 in a yield of more than 30% of the total products from short-chain amylose (DP = 17). The crystal structures revealed that Asp236 is a nucleophilic catalyst and Glu266 is a proton donor/acceptor. Pseudo-maltononaose occupies subsites -6 to +3 and induces the conformational change of Glu266 and Asp333 to form a salt linkage with the N-glycosidic amino group and a hydrogen bond with secondary hydroxyl groups of the cyclitol residue bound to subsite -1, respectively. The indole moiety of Trp140 is stacked on the cyclitol and 4-amino-6-deoxyglucose residues located at subsites -6 and -5 within a 4 A distance. Such a face-to-face short contact may regulate the disposition of the glucosyl residue at subsite -6 and would govern the product specificity for G6 production.
Kanai
Ryuta
R
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Haga
Keiko
K
Akiba
Toshihiko
T
Yamane
Kunio
K
Harata
Kazuaki
K
D000688:Amylose / Q000737:chemistry / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000378:metabolism*
D005936:Glucans; D009844:Oligosaccharides; C021705:maltooligosaccharides; C019193:maltopentaose; C016549:maltohexaose; C031146:maltoheptaose; C101184:maltononaose; D000688:Amylose; D000520:alpha-Glucosidases; C065485:glucan 1,4-alpha-maltohexaosidase