tailieunhanh - Báo cáo khoa học: The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II (TVA II) complexed with transglycosylated product

Ana-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzesa-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzesa-1,4-glucosidic linkages of cyclodextrins anda-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4 3 -a-panosylpanose (4 3 -P2) (a pullulan hydro-lysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. | Eur. J. Biochem. 271 2530-2538 2004 FEBS 2004 doi The crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II TVA II complexed with transglycosylated product Masahiro Mizuno1 Takashi Tonozuka1 Akiko Uechi1 Akashi Ohtaki2 Kazuhiro Ichikawa1 Shigehiro Kamitori2 Atsushi Nishikawa1 and Yoshiyuki Sakano1 1Departments of Applied Biological Science and 2Biotechnology and Life Science Tokyo University of Agriculture and Technology Tokyo Japan An a-amylase TVA II from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes a-1 4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes a-1 4-glucosidic linkages of cyclodextrins and a-1 6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II we soaked 43-a-panosylpanose 43-P2 a pullulan hydrolysate composed of two panosyl units into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 42-a-pano-sylpanose 42-P2 which was produced by transglycosylation from 43-P2 at resolution. The shape of the active cleft of TVA II is unique among those of a-amylase family enzymes due to a loop residues 193-218 that is located at the end of the cleft around the nonreducing region and forms a dam -like bank. Because this loop is short in TVA II the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc 2 of the substrate it appears that Tyr374 in proximity to Trp356 plays two roles one is fixing the orientation of Trp356 in the substrate-li-ganded state and the other is supplying the water that is necessary for substrate hydrolysis. Keywords a-amylase GH family 13 42-a-panosylpanose substrate specificity transglycosylation. a-Amylase 1 4-a-D-glucan-4-glucanohydrolase EC hydrolyzes

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