A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi.
Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 degrees C and pH 6.5. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in lambdaZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A") was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 degrees C and a pH optimum of 6.5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A" cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A") bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A" consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.
Halstead
Jonathan R
JR
Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK1.
Vercoe
Philip E
PE
Department of Animal Science, University of Western Australia, Nedlands, WA 6009, Australia2.
Gilbert
Harry J
HJ
Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK3.
Davidson
Keith
K
Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK1.
Hazlewood
Geoffrey P
GP
Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK1.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002482:Cellulose / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000821:virology; D004926:Escherichia coli / Q000235:genetics; D005658:Fungi / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D016415:Sequence Alignment; D013696:Temperature; D044902:beta-Mannosidase
D008351:Mannans; C012990:galactomannan; D002482:Cellulose; D008361:Mannosidases; D044902:beta-Mannosidase; D005690:Galactose