Characterization of a beta-N-acetylhexosaminidase and a beta-N-acetylglucosaminidase/beta-glucosidase from Cellulomonas fimi.
The gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular beta-N-acetylglucosaminidases, a family 20 beta-N-acetylhexosaminidase (Hex20), and a novel family 3-beta-N-acetylglucosaminidase/beta-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 degrees C were 0.066 s(-1) x mM(-1) and 0.076 s(-1) x mM(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 4'-nitrophenyl beta-D-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between beta-N-acetylglucosaminidases and beta-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl-enzyme species trapped with the slow substrate 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave beta-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the D-glucosyl/D-galactosyl configuration of the glycone: Km and kcat values were 53 microM and 482.3 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 66 microM and 129.1 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-galactosaminide.
Mayer
Christoph
C
Department of Chemistry, University of British Columbia, Vancouver, Canada. ch.mayer@uni-konstanz.de
Vocadlo
David J
DJ
Mah
Melanie
M
Rupitz
Karen
K
Stoll
Dominik
D
Warren
R A J
RA
Withers
Stephen G
SG
D000118:Acetylglucosaminidase / Q000737:chemistry*; D000595:Amino Acid Sequence; D040143:Cellulomonas / Q000201:enzymology*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry*; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry*
D001617:beta-Glucosidase; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases