Structure and function of the type III pullulan hydrolase fromThermococcus kodakarensis

Abstract

Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95-100°C and has a pH optimum in the range 3.5-4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine.

Understanding Thermostability Factors of Barley Limit Dextrinase by Molecular Dynamics Simulations

“…Salt bridges in enzyme may contribute to its stability at high temperature (Vieille and Zeikus, 2001;Kundu and Roy, 2010). Recently, Guo et al (2018) summarized factors may contribute to the thermostability for pullulan-hydrolyzing enzymes. They found that there are more salt bridges in thermophilic pullulan-hydrolyzing enzymes than mesophilic ones, suggesting the importance of salt bridge for the enzyme pullulan-hydrolyzing thermostability.…”

TK-PUL, a pullulan hydrolase type III from Thermococcus kodakarensis, a potential candidate for simultaneous liquefaction and saccharification of starch

“…TIM-barrel) type catalytic domain; (iii) their primary structures contain the four highly conserved sequence regions; and (iv) their catalytic triad is constituted by the three acidic residues (two aspartic acids and one glutamic acid). Biochemical characterization and three-dimensional structure analysis of TK-PUL revealed that it possessed all the features of the family GH13 [3,8].…”

“…Because of this novel domain, monomers of archaeal enzymes remain successful to adopt a unique active-site configuration for cyclodextrinase activity [24,25]. Tertiary structure of TK-PUL [8] was found similar with the structures of other archaeal members of the subfamily GH13_20, which are maltogenic amylase from Staphylothermus marinus [24] and α-amylase/cyclomaltodextrinase from Pyrococcus furiosus [25]. TK-PUL lacks significant homology with these enzyme at the N-terminal region and probably due to this reason TK-PUL, in spite of high cyclodextrinase activity [3], is not a member of either CBM34 or CBM48 (http://www.cazy.org/).…”

“…In order to improve yields of maltose and glucose syrups, pullulanase is employed as a debranching enzyme, which hydrolyses α-1,6-glucosidic linkages at the branch points of limit dextrins. Both α-and β-amylases are unable to hydrolyze α-1,6 bonds [8]. On the basis of sequence similarities pullulanases are grouped into glycoside hydrolase (GH) families GH13 and GH57 [9,10] and, eventually, also in the family GH49 (http://www.…”

“…Moreover, it does not require any additional metal ion for its activity or stability [3]. Three-dimensional structure of TK-PUL has already been resolved [8]. Like other members of GH13, TK-PUL is a multi-domain enzyme consisting of an N-terminal domain (residues 185-280), a central catalytic domain (residues 281-694) in the form of typical TIM-barrel structure and a C-terminal domain (residues 694 onwards).…”