Protein thermostability has been investigated by two approaches. (A) Computational. To study the relationship between thermostability and conformational characteristics of proteins, 195 single amino acid residue replacements have been analysed for several protein conformational characteristics. From the analyses, some general rules arise which suggest where amino acid substitutions can be made to enhance protein thermostability. (B) Experimental. Glucohydrolases are biotechnologically important enzymes. We are analysing by site directed mutagenesis the structure/function relationship of two bacterial glucohydrolases, a 1,3-1,4-beta-glucauase and a beta-glucosidase. We have determined the key residues for catalysis and substrate binding, and redesigned the stability and specificity of the glucanase. A glucanase thermorresistant mutant (N57A) has been obtained.

PCR methodology is one of the fastest available procedures for site-directed mutagenesis (1,2). However, it has been criticized for a lack of reliability because of unwanted mismatches produced during the PCR reaction (3,4). In the present protocol, we describe an improvement on the efficiency of site-directed mutagenesis by PCR using the Pyrococcus species GB-D polymerase instead of the commonly used Thermus aquatiqus (Taq) polymerase. Taq polymerase lacks a 3′→5′ proofreading exonuclease activity that is not crucial for several PCR applications, but is advisable for site-directed mutagenesis experiments. Some thermophilic DNA polymerases have this activity, among them the Thermococcus litoralis and the Pyrococcus species GB-D enzymes. A 10-fold higher efficiency has been reported for these enzymes over that observed for Taq polymerase (5). PCR site-directed mutagenesis is specially suitable for protein engineers when it is coupled to a screening procedure directly performed on the transformant plates. In such cases the procedure is rapid (3 d from mutagenic primers to selection of clones) and efficient (98–100% of successful mutagenesis).

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1,4-β-d-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to β-strand-(87–95) of the minor β-sheet III of the protein core by a single disulfide bond at Cys61–Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The ΔΔG Ubetween oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 ± 0.2 kcal·mol−1 for the disulfide bridge to protein stability. A good correlation is observed between ΔG U values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase inK m correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (k cat/K m) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with K m > 2-fold andk cat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in k cat), but it is slightly less stable.

Bacillus 1,3-1,4-beta-glucanases possess a highly conserved disulfide bridge connecting a beta-strand with a solvent-exposed loop lying on top of the extended binding site cleft. The contribution of the disulfide bond and of both individual cysteines (Cys61 and Cys90) in the Bacillus licheniformis enzyme to stability and activity has been evaluated by protein engineering methods. Reduction of the disulfide bond has no effect on kinetic parameters, has only a minor effect on the activity-temperature profile at high temperatures, and destabilizes the protein by less than 0.7 kcal/mol as measured by equilibrium urea denaturation at 37 degrees C. Replacing either of the Cys residues with Ala destabilizes the protein and lowers the specific activity. C90A retains 70% of wild-type (wt) activity (in terms of Vmax), whereas C61A and the double mutant C61A-C90A have 10% of wt Vmax. A larger change in free energy of unfolding is seen by equilibrium urea denaturation for the C61A mutation (loop residue, 3.2 kcal/mol relative to reduced wt) as compared with the C90A mutation (beta-strand residue, 1.8 kcal/mol relative to reduced wt), while the double mutant C61A-C90A is approximately 0.8 kcal/mol less stable than the single C61A mutant. The effects on stability are interpreted as a result of the change in hydrophobic packing that occurs upon removal of the sulfur atoms in the Cys to Ala mutations.

The crystal structure of the 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 Å and refined to R = 16.5%. The protein has a similar β‐sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16‐M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3‐1,4‐β‐d‐glucan 4‐glucanohydrolases.

Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.