X-ray crystallographic analysis of type I pullulanases fromKlebsiella pneumoniaeandBacillus subtilis

Abstract

Pullulanase (EC 3.2.1.41) catalyzes the hydrolysis of α-1,6glycosidic linkage ofα-glucans such as amylopectin and pullulan. The enzyme is classified to family 13 of glycoside hydrolase together with α-amylase and isoamylase. In order to elucidate the enzymatic mechanism of pullulanase, we have determined the crystal structures of the enzyme from Klebsiella aerogenes (KPP) and Bacillus subtilis (BSP) by M.I.R and MR, resspectively. KPB structures of apo and complexes with glucose (G1), maltose (G2), matotriose (G3), matotetraose (G4) and isomaltose (isoG2) were refined at 1.6-1.9 A resolution. BSP structures of apo and complexes with G2, and α-cyclodexrin (αCD) were refined at 2.1-2.3 A resolution. KPP is consist of five domains (N1, N2, N3, A and C), while BSP has four domains lacking N1 domain of KPP, which is a new type of starchbinding domain with one calcium site (CBM41). In catalytic A domain, BSP has shorter loops (res. 446-559, 771-783 and 913-945 in KPP are deleted) and resembles to isoamylase [1] more than KPP except for a few loops characteristics to both enzymes. The subsite mapping of KPP by the binding of the oligosaccharides clearly showed the parallel binding of two oligosaccharides in the active site (subsites +2∼-1’ and -1∼-4). The catalytic acid (Glu706 in KPP and Glu435 in BSP) and nucleophile (Asp677 in KPP and Asp406 in BSP) are situated at the junction of the two sugar binding sites (between +1 and -1). One αCD was found to bind in the edge of the active cleft of BSP around subsite +2 of the oligosaccharide-binding site. The side chain of Phe476, which is in the pullulanase specific loop, occupied the hole of αCD from its hydrophobic side. These findings provide an important structural evidence to account the different enzymatic properties between pullulanase and isoamylase.