Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11

Abstract

Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg(-1) and 16.4 μmol L(-1), respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75-2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain.

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

“…These C-terminal tandem domains assume an immunoglobulin-like fold typical for starch binding domains, and show close structural resemblance (but low sequence identity ~ 18%) to the N-terminal domains (N1 and N2) of Anoxybacillus sp . LM18-11 pullulanase [35], with N1 (10–88) corresponding to TarS C2 (overall main chain rmsd of 1.0 Å over 25 atom pairs) and N2 (110–186) corresponding to TarS C1 (overall main chain rmsd of 1.2 Å over 36 atom pairs). Interestingly, the tandem domains of the two enzymes are inverted with respect to their corresponding catalytic domains, existing C-terminally in TarS and N-terminally in pullulanase.…”

Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

“…interactions is absent in the PULs from GH13_12 [21,22] and GH13_14 [23][24][25]. It is likely that this dynamic Phe620-Asp621 loop is important for HvLD activity on different length limit dextrins during amylopectin conversion.…”

“…The KpPUL has an extra N-terminal carbohydrate binding module compared to its plant counterparts. Additionally two structures of Streptococci PULs from GH13_12 [21,22] and three structures of Bacilli PULs from GH13_14 are known [23][24][25]. None of the debranching enzyme structures published to date have been substrate complexes, i.e.…”

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

“…It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, ␤-amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2,3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4,5).…”