Article Figures & Data
Figures
- Fig. 1.
Determination of the Mr, pI, and structural composition of purified CelF by analytical PAGE. (A) Samples from each of the five stages of enzyme purification were denatured, resolved by SDS-PAGE (4 to 12% acrylamide; Bis-Tris gel), and stained with Coomassie brilliant blue. Lane 1, high-speed supernatant; lane 2, DEAE Tris-Acryl M; lane 3, phenyl-Sepharose CL-4B; lane 4, Ultrogel AcA-44; and lane 5, purified CelF (Mr, ∼50,000; ∼8 μg) from Blue Tris-Acryl. Also shown are Novex Mark 12 molecular weight markers (Std). (B) Determination of the pI of CelF (∼4.9) by analytical electrofocusing. Approximately 1 μg of enzyme was applied directly to the surface of the isoelectric focusing gel in lane 2 and protein standards (pI range, 3.5 to 9.3; Pharmacia) in lanes 1 and 3. (C) Cross-linking of the subunits of native CelF by dimethyl adipimidate (DMA). A mixture containing ∼40 μg of CelF and ∼200 μg of DMA was prepared in 20 μl of 0.1 M triethanolamine buffer (pH 8). After 2 h of incubation at room temperature, 10 μl of the sample was denatured and analyzed by SDS-PAGE (lane 3). M, D, T, and Tet, monomer, dimer, trimer, and tetrameric species, respectively. Denatured monomer (control; no DMA) and molecular weight markers are shown in lanes 1 and 2, respectively.
- Fig. 2.
Unrooted neighbor-joining phylogenetic tree of family 4 glycosylhydrolases. The 16 protein sequences were aligned by the program CLUSTAL X (41, 42). For pairwise alignments, a gap-opening penalty of 35 and a gap extension penalty of 0.75 were used. For multiple alignments, a gap-opening penalty of 15 and a gap extension penalty of 0.3 were used. The alignment was used to construct the neighbor-joining tree (33) as implemented by PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b1a (37, 38). The numbers are bootstrap values, and branch lengths indicate the fraction of amino acid substitutions, as indicated on the scale. Proteins that have been purified to homogeneity and characterized are boxed. Entries without gene or protein names (E. faecalis, V. cholerae, etc.) are derived from sequences of unfinished genomes.
- Fig. 3.
Alignment of the amino acid sequences of the three enzymes from family 4 that have been purified and characterized: CelF (P-β-glucosidase from E. coli [this study]), MalH (P-α-glucosidase; F. mortiferum [5]), and GlvA (P-α-glucosidase; B. subtilis [40]). Identical residues are highlighted, and putative catalytic residues are marked by asterisks. The numbers to the left and right denote residue positions, and the numbers above the residues refer to alignment positions.
Tables
- Table 1.
Metal ion specificity and NAD+ requirement for activity of CelF
Addition to assaya Specific activityb Control (no addition) 0.017 Mn2+ 0.249 Mn2+ + NAD+ 1.155 Ni2+ 0.042 Ni2+ + NAD+ 0.880 Co2+ 0.197 Co2+ + NAD+ 0.292 Mg2+ NDc Mg2+ + NAD+ 0.065 Sr2+ ND Sr2+ + NAD+ 0.061 Ca2+ ND Ca2+ + NAD+ 0.048 ↵a The 2-ml assay mixture contained 50 mM Tris-HCl buffer (pH 7.5) and, when required, 1 mM appropriate divalent metal ion, 1 mM NAD+, and 0.38 mg of dialyzed CelF. After 5 min of preincubation at 37°C, the substrate (pNPβG6P) was added to a final concentration of 1 mM.
↵b Expressed as micromoles of pNPβG6P hydrolyzed minute−1 milligram of enzyme−1.
↵c ND, no detectable activity.
- Table 2.
Substrate specificity and kinetic parameters of purified CelF
P-β-glucoside Km(mM) Vmax (μmol min−1mg−1) 4MUβG6P 0.30 ± 0.02 3.35 ± 0.08 Arbutin-6Pa 0.35 ± 0.04 2.13 ± 0.08 pNPβG6P 0.44 ± 0.04 1.33 ± 0.05 Salicin-6Pb 0.44 ± 0.09 1.38 ± 0.09 Cellobiose-6P 1.30 ± 0.09 1.74 ± 0.05 MethylβG6P 2.22 ± 0.21 0.56 ± 0.03 Gentiobiose-6Pc 12.50 ± 1.26 1.11 ± 0.11
