Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equi

• Published in: Microbiology (Society for General Microbiology) Vol. 146; no. 8; pp. 1999 - 2008
• Main Authors: Eberhardt, Ruth Y, Gilbert, Harry J, Hazlewood, Geoffrey P
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.2000; Society for General Microbiology
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Catalytic Domain - genetics; Cel45A protein; Cel5A protein; Cellulase - chemistry; Cellulase - genetics; Cellulase - metabolism; DNA Primers - genetics; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; endoglucanase; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Hydrogen-Ion Concentration; Microbiology; Molecular Sequence Data; Molecular Weight; Mycology; Piromyces - enzymology; Piromyces - genetics; Piromyces equi; Protein Structure, Tertiary - genetics; Sequence Homology, Amino Acid; Substrate Specificity; Fungi; Enzymatic activity; Cellulolytic enzyme; Nucleotide sequence; Enzyme; Glycosidases; Substrate specificity; Hydrolases; O-Glycosidases; Aminoacid sequence; Thallophyta; Glucanase
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-146-8-1999

Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK 1 Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK 2 Author for correspondence: Ruth Y. Eberhardt. Tel: +44 1223 496478. Fax: +44 1223 496023. e-mail: ruth.eberhardt{at}bbsrc.ac.uk Two endoglucanase cDNAs, designated cel5A and cel45A , were isolated from a cDNA library of the anaerobic fungus Piromyces equi . Sequence analysis revealed that cel5A has an open reading frame of 5142 bp and encodes a 1714 amino acid modular enzyme, Cel5A, with a molecular mass of 194847 Da. Cel5A consists of four catalytic domains homologous to family-5 glycosyl hydrolases, two C-terminal dockerins and one N-terminal dockerin. This is the first report of a complete gene containing tandem repeats of family-5 catalytic domains. The cDNA cel45A has an open reading frame of 1233 bp and encodes a 410 amino acid modular enzyme, Cel45A, with a molecular mass of 44380 Da. The catalytic domain, located at the C terminus, is homologous to the family-45 glycosyl hydrolases. Cel45A is the first family-45 enzyme to be described in an anaerobe. The presence of dockerins at the N and C termini of Cel5A and at the N terminus of Cel45A implies that both enzymes are part of the high-molecular-mass cellulose-degrading complex produced by Piromyces equi . The catalytic domain nearest the C terminus of Cel5A and the catalytic domain of Cel45A were hyperexpressed as thioredoxin fusion proteins, Trx-Cel5A' and Trx-Cel45A', and subjected to biochemical analysis. Trx-Cel5A' has a broad substrate range, showing activity against carboxymethylcellulose, acid-swollen cellulose, barley ß-glucan, lichenin, carob galactomannan, p -nitrophenyl ß-D-cellobiopyranoside and xylan. Trx-Cel45A' is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley ß-glucan and lichenin. Keywords: anaerobic fungi, cellulase, glycosyl hydrolases (families 5 and 45), endoglucanases, Piromyces equi Abbreviations: CMC, carboxymethylcellulose; CMCase, carboxymethylcellulase; DNSA, dinitrosalicylic acid reagent; pNPC, p -nitrophenyl ß-D-cellobiopyranoside The EMBL accession numbers for the sequences reported in this paper are AJ277482 and AJ277483. a Present address: Finnfeeds International, PO Box 777, Marlborough SN8 1XN, UK.