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 specidegrees 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 thed-glucosyl/d-galactosyl configuration of the glycone: K m and k cat values were 53 micro m and 482.3 s-1 for 4'-nitrophenyl beta-N -acetyl-d-glucosaminide and 66 micro m and 129.1 s-1 for 4'-nitrophenyl beta-N -acetyl-d-galactosaminide.PUBLICATION ABSTRACT