An extended loop in CE7 carbohydrate esterase family is dispensable for oligomerization but required for activity and thermostability

Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7

“…The pairwise sequence identities of the full set of sequences are in the range 17–98%, indicating significant levels of sequence divergence within the family. The structures of TmAcE and its complex with a noncognate substrate analog 2‐(2‐oxo‐1,3‐dihydroindol‐3‐yl)acetate (TmAcE‐OIA) determined at 1.76 and 1.86 Å resolution, respectively, were reported earlier (PDB: 5FDF, 5JIB) . Using the OIA complex structure as the reference, the multiple sequence alignment was examined for conservation patterns of residues that constitute the active site and the structural environment of the cis Pro228 residue.…”

“…The mutation was confirmed by DNA sequencing of the entire protein coding region of the purified plasmid. The expression and purification of the mutant protein was identical to the previously described protocol followed for the wild type TmAcE . Purified protein in the final buffer containing 10 m M Tris HCl, pH 8.0, 150 m M NaCl was concentrated to 20 mg mL −1 and stored at −40°C.…”

“…The asymmetric unit contained one hexamer. The isomorphous monoclinic crystal form of wild type TmAcE (PDB: 5FDF) was directly used as a model for refinement. To minimize model bias, residues in the range 224–232 around the cis Pro228 residue were deleted from the model prior to refinement.…”

“…The catalytic activities of the wild type and the mutant were measured spectrophotometrically using six substrates including generic esters, p ‐nitrophenyl acetate ( p NPA), 4‐methylumbelliferyl acetate (MUFA), 1‐naphthyl acetate (1‐NA), and specific substrates, glucose penta‐acetate (5‐Ac‐Glu), xylose tetra‐acetate (4‐Ac‐Xy), and 7‐ACA according to the procedure described previously . Measurements were made in a Perkin Elmer Lambda 25 UV visible spectrometer equipped with a Peltier system.…”

“…To date, crystal structures of four CE7 orthologs from Thermotoga maritima , Bacillus subtilis , Thermoanerobacterium JW/SL YS485, and Bacillus pumilus PS2 have been determined. The tertiary structure displaying the canonical α/β hydrolase fold consists of a central eight‐stranded β‐sheet flanked by α‐helices on either side, with the active site containing a highly conserved geometry of the catalytic Ser‐His‐Asp triad and oxyanion hole interactions …”