Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707.
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.
Kanai
Ryuta
R
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.
Haga
Keiko
K
Akiba
Toshihiko
T
Yamane
Kunio
K
Harata
Kazuaki
K
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D001467:Hordeum / Q000201:enzymology; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008024:Ligands; D009844:Oligosaccharides; C019193:maltopentaose; C016549:maltohexaose; D014364:Tryptophan; D000520:alpha-Glucosidases; C065485:glucan 1,4-alpha-maltohexaosidase