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Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase.

ABSTRACT: CBMs (carbohydrate-binding modules) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain approx. 100 amino acid residues, and some members have starchbinding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the SBD (starch-binding domain) of Rhizopus oryzae glucoamylase (RoCBM21) determined by NMR spectroscopy. This CBM has a beta-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analysed by chemical-shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, namely type I and type II, with CBM20, CBM25, CBM26 and CBM41 showing type I topology, with CBM21 and CBM34 showing type II topology. According to the chemical-shift perturbations, RoCBM21 contains two ligand-binding sites. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is embedded in a region with unique sequence motifs only found in some members of CBM21s. Additionally, docking of beta-cyclodextrin and malto-oligosaccharides highlights that side chains of Y83 and W47 (one-letter amino acid code) form the central part of the conserved binding platform in the SBD. The structure of RoCBM21 provides the first direct evidence of the structural features and the basis for protein-carbohydrate recognition from an SBD of CBM21.

SUBMITTER: Liu YN

PROVIDER: S-EPMC1828892 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase.

Liu Yu-Nan YN Lai Yen-Ting YT Chou Wei-I WI Chang Margaret Dah-Tsyr MD Lyu Ping-Chiang PC

The Biochemical journal 20070401 1

CBMs (carbohydrate-binding modules) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain approx. 100 amino acid residues, and some members have starchbinding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the SBD (starch-binding domain) of Rhizopus oryzae glucoamylase (RoCBM21) determined by NMR spectroscopy. This CBM has a beta-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of R ...[more]

PMID: 17117925

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Project description:BackgroundGlucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost.ResultsReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4-5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch.ConclusionsOur results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Dataset Information

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase.

Publications

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase.

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