Mannan-Degrading Enzymes from Cellulomonas fimi

Abstract

The genes man26a and man2A fromCellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced byEscherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.

-Mannosynthase: Synthesis of -Mannosides with a Mutant -Mannosidase

“…to yield a 95 kDa monomeric enzyme belonging to family 2 of the glycosyl hydrolases. [11] The active site nucleophile was identified as Glu 519 by trapping the covalent mannosyl ± enzyme intermediate with the mechanism-based inactivator 2-deoxy-2-fluoro b-d-mannosyl fluoride, followed by proteolysis and sequencing of the labeled peptide by ESI-MS/MS. [12,13] Mutation of the nucleophile to alanine (Glu 519 Ala) produced an enzyme that was inactive as a hydrolase.…”

Phylogenetic analysis and substrate specificity of GH2 β‐mannosidases from Aspergillus species

“…This contrasts MndA, which previously has been shown to release mannose from GM 2 and G 2 M 5 ([2], Table 1). MndB, MndA and the known intracellular b-mannosidase from C. fimi (CfMan2A) [2] were incubated with INM and analysed for mannose release. MndA released 11% mannose in 24 h, while no mannose release was detected for MndB (Table 1) and CfMan2A.…”

“…b-Mannosidases have been reported from both bacteria [1,2] and fungi [3][4][5][6]. They are also present in plants and animals, including mammals [7][8][9] and are mainly classified into glycoside hydrolase family 2 (GH2), a diverse family with several exo-glycosidase specificities.…”

“…b-Mannosidases have been characterised from various environments. For instance, the soil bacterium Cellulomonas fimi produces an intracellular GH2 b-mannosidase (CfMan2A) [2], while the human gut bacterium Bacteroides thetaiotamicron secretes a GH2 b-mannosidase (BtMan2A) [12]. BtMan2A is also hitherto the only high-resolution GH2 b-mannosidase crystal structure, showing an active site pocket with a À1 subsite essential for recognition of terminal mannosyl residue [12].…”

“…Given the fact that b-mannosidases are widely distributed and b-mannoside structures occur in various types of substrates, these enzymes may have evolved to hydrolyse different substrates in quite different cellular locations and environments. b-Mannosidase activity is widely assigned based on the hydrolytic activity on artificial colorimetric substrates such as p-nitrophenyl-b-mannopyranoside (pNP-b-Man) [1,2]. Such analysis, however, does not give any indication about differences in fine-tuned substrate specificity, for example, the effect of substrate degree of polymerization or substitutions carried by mannosyl moieties.…”