Characterization of a β-N-acetylhexosaminidase and a β-N-acetylglucosaminidase/β-glucosidase from Cellulomonas fimi

The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β-N-acetylglucosaminidases, a family 20 β-N-acetylhexosaminidase (Hex20), and a novel family 3-β-N-acetylglucosaminidase/β-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: k_cat/K_m values at 25 °C were 0.066 s⁻¹·mm⁻¹ and 0.076 s⁻¹·mm⁻¹ for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 4′-nitrophenyl β-d-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β-N-acetylglucosaminidases and β-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-β-d-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave β-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: K_m and k_cat values were 53 µm and 482.3 s⁻¹ for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 66 µm and 129.1 s⁻¹ for 4′-nitrophenyl β-N-acetyl-d-galactosaminide.