Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli

doi:10.1016/j.pep.2004.05.008

Protein Expression and Purification

Volume 37, Issue 1, September 2004, Pages 170-179

https://doi.org/10.1016/j.pep.2004.05.008 Get rights and content

Abstract

The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E. coli were purified and characterized. YicI has the activity of α-xylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7–10.1 with incubation for 24 h at 4 °C and also is stable up to 47 °C with incubation for 15 min. The enzyme shows higher activity against α-xylosyl fluoride, isoprimeverose (6-O-α-xylopyranosyl-glucopyranose), and α-xyloside in xyloglucan oligosaccharides. The α-xylosidase catalyzes the transfer of α-xylosyl residue from α-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against α-glucosyl fluoride, and so is an α-glucosidase, although the natural substrates, such as α-glucobioses, are scarcely hydrolyzed. α-Glucosidase has been found for the first time in E. coli.

Section snippets

Substrates

To obtain a xyloglucan oligosaccharide mixture (XGOS), tamarind seed polysaccharide (Tokyo Kasei Kogyo, Tokyo, Japan) was digested with Trichoderma viride cellulase (Sigma, St. Louis, MO, USA) and debris was removed by centrifugation (20,000g for 10 min). Ethanol was added to the supernatant up to 60%, followed by centrifugation of the suspension. XGOS was prepared from the supernatant [15]. Isoprimeverose (6-O-α-xylopyranosyl-glucopyranose) was obtained by treating tamarind seed polysaccharide

Sequence analysis of yicI and yihQ

The similarity of the amino acid sequences of YicI and YihQ was investigated by using BLASTP program in Swiss-Prot/TrEMBL database. The high score homolog, whose enzymatic function has become apparent, is shown sequentially from the top to 10 Table 1. YicI exhibits the similarity to α-xylosidases, α-glucosidases, and isomaltosyltransferase, especially 43% identity with Lactobacillus pentosus α-xylosidase. YihQ exhibits the similarity to Bacillus thermoamyloliquefaciens α-glucosidase III and II,

Discussion

A number of glycosidase amino acid sequences have been unveiled by genome sequencing project and classified into GH families on the basis of their structural features [1], [2], [3]. GH family 31 includes various types of enzymes, such as α-glucosidase, sucrase-isomaltase, α-xylosidase, α-glucan lyase, and isomaltosyltransferase, from archea, bacteria, and eukaryote. However, it is ambiguous as to whether most of the proteins are substantially α-glucosidase, α-xylosidase or the others, because

Acknowledgements

We thank Dr. M. Kitaoka in National Food Research Institute, who kindly taught us synthesis skill of glycosyl fluorides. We are grateful to Mr. T. Hirose and Mr. Y. Abe in the Centre for Instrumental Analysis, Hokkaido University, for amino acid analysis and Dr. E. Fukushi in the GC-MS & NMR Laboratory, Graduate School of Agriculture, Hokkaido University, for measuring the NMR. A part of this work was financially supported by Grant-in-Aid Scientific Research from The Japan Society of Promotion

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We suggest that the reorientation of this catalytic residue together with the constriction of xylosyl binding site highlight the structural changes that explain the experimentally observed reduction in the catalytic efficiency of the D49A mutant (Fig. 4 and Table 1). The α-xylosidase activity of YicI, removes the α-xylosyl moieties at the non-reducing end of XGOs (Lovering et al., 2005; Okuyama et al., 2004), and in addition can also hydrolyze the α(1,6) glycosidic linkage in isoprimeverose (Xyl-α(1,6)-Glc) (Okuyama et al., 2004). X-ray crystallography and 5-fluoroglycosyl fluoride trapping experiments have provided evidence supporting a retaining Koshland double-displacement mechanism in which the D416 acts as the nucleophile and the D482 as the general acid/base residue (Lovering et al., 2005).
Xyloglucan is ubiquitous in the cell walls of land plants and is also an essential storage polymer in seeds of many species. We studied the hydrolysis of the non-reducing end xylosyl residue of xyloglucan oligosaccharides (XGOs) by the Escherichia coli α-xylosidase (YicI). Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and ion fragmentation analysis together with high performance anion exchange chromatography with pulsed amperometric detection revealed that YicI preferentially removes the xylosyl residue from the glycosyl residue of non-galactosylated oligosaccharides. The YicI shows decreasing activity against the galactosylated oligosaccharides XXXG>XXLG≥XLXG. Studies of the XGOs interaction with active site residues by molecular dynamics simulations suggested that hydrogen bond interactions between the D49 and galactosylated oligosaccharides play an important role in enzyme-XGO interactions. This was confirmed by site-directed mutagenesis, where the D49A mutant affected catalytic efficiency against galactosylated XGOs. Our findings advance xyloglucan disassembly models and highlight the importance of YicI for biotechnology applications.
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In contrast, previously reported α-xylosidases, including S. solfataricus XylS (18), T. majus Q9ZP04 (48) and E. coli YicI (19), prefer isoprimeverose as a substrate over pNP α-d-xylopyranoside, indicating that the substrate specificity of MeXyl31 differs from that of other α-xylosidases. In addition, although most α-xylosidases, such as S. solfataricus XylS (18), T. majus Q9ZP04 (48) and E. coli YicI (19), exhibit α-glucosidase activity toward maltose, α-glucosidase activity of recombinant MeXyl31 toward maltose was not detected. Crystal structures of α-xylosidase, E. coli YicI (41), and C. japonicus Xyl31A (40) have been elucidated previously.
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Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli

Abstract

Section snippets

Substrates

Sequence analysis of yicI and yihQ

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Anal. Biochem.

Anal. Biochem.

Biochim. Biophys. Acta

FEBS Lett.

Carbohydr. Res.

A classification of glycosyl hydrolases based on amino acid sequence similarities

Biochem. J.

New families in the classification of glycosyl hydrolases based on amino acid sequence similarities

Biochem. J.

Updating the sequence-based classification of glycosyl hydrolases

Biochem. J.

The complete genome sequence of Escherichia coli K-12

Science

Plant α-glucosidases of the glycoside hydrolase family 31. Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin

Plant Mol. Biol.

Molecular mechanism in α-glucosidase and glucoamylase

Biosci. Biotechnol. Biochem.

Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of α-glucosidase from Schizosaccharomyces pombe

Eur. J. Biochem.

Substrate specificity of Bacillus α-d-xylosidase

Agric. Biol. Chem.

Isolation of Aspergillus flavus MO-5 producing two types of intracellular α-d-xylosidases: purification and characterization of α-d-xylosidase I

Biosci. Biotechnol. Biochem.

Classification of some α-glucosidases and α-xylosidases on the basis of substrate specificity

Biosci. Biotechnol. Biochem.

Transfer action of α-d-xylosidases from Aspergillus flavus MO-5 on p-nitrophenyl-α-d-xylopyranoside

Biosci. Biotechnol. Biochem.