David A. Estell

Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin

Structural studies on serine proteinases have shown that hydrogen bonds are involved in stabilizing the charged tetrahedral intermediate in the transition-state complex. However, little is known about the quantitative contribution of these interactions to transition-state stabilization. X-ray crystallographic studies of subtilisin (Robertus, J. D., Kraut, J., Alden, R. A. & Birktoft, J. J. Biochemistry, Wash. 11, 4293-4303 (1972)) have suggested that the amide side chain from asparagine-155 forms a hydrogen bond with the oxyanion produced on the substrate carbonyl oxygen in the tetrahedral intermediate. To study the importance of the Asn-155 hydrogen bond in stabilizing the tetrahedral intermediate, Asn-155 was substituted with Thr, His, Gin and Asp by using site-specific mutagenesis of the cloned subtilisin gene from B. amyloliquefaciens. These substitutions were intended to alter the position and charge of the potential hydrogen-bonding group at 155. Mutations of Asn-155 caused large decreases in substrate turnover, Kcat (200- to 4000-fold), with marginal decreases in substrate binding, KM (up to 7-fold). The most dramatic effects were seen with Thr-155, where Kcat was reduced 4000-fold with a slight increase in KM. Mutations of Asn-155 caused a loss in transition-state stabilization energy of 9.2-20 kJ mol-1 Simple enrichment methods are described which greatly facilitate the isolation of mutant sequences. These methods depend upon the introduction or elimination of a unique and silent restriction site near the site of mutagenesis.

Subtilisin — an enzyme designed to be engineered

Abstract Almost every property of subtilisin, a simple bacterial serine protease, has been altered by protein engineering including its catalysis, substrate specificity, pH/rate profile, and stability to oxidative, thermal and alkaline inactivation.

Probing Steric and Hydrophobic Effects on Enzyme-Substrate Interactions by Protein Engineering

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly166), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (kcat/Km) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft (∼160 �3), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (kcat/Km) (up to 5000 times) as a result of steric hindrance.

Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modffied Erwinia herbicola

A new metabolic pathway has been created in the microorganism Erwinia herbicola that gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, a Corynebacterium enzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from a Corynebacterium genomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to the Escherichia coli trp promoter and a synthetic ribosome binding site and was then introduced into E. herbicola on a multicopy plasmid. Erwinia herbicola naturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.

Engineering enzymes for improved performance in industrial applications

Abstract Four enzymes of industrial importance have been engineered for improved properties in commercial applications. Two different subtilisins have been engineered to give greater cleaning efficiency in detergent applications. A lipase from Pseudomonas has been altered in order to allow it to catalyze the synthesis of peracid bleaches. The β-galactosidase from Lactobacillus has been engineered to lose activity at 4°C in order to increase the storage shelf life of yogurt. These examples indicate that protein engineering is being successfully applied to a number of industrial applications.

Altering the proteolytic activity of subtilisin through protein engineering.

The utility of protein engineering in redesigning the structure of a protein to tailor its functional properties has been firmly established. In particular, the Bacillus serine protease subtilisin has proven to be a useful model protein for examining the use of systematic structural modification to incorporate novel functional properties into an enzyme.1.2 The list of properties that have been altered in subtilisin via such modification includes oxidative ~tability,”~ thermal ~tability,~ alkaline pH stability,h stability in organic ~olvent ,~ substrate specificity in aqueous nucleophile specificity,l2.l3 and pH activity profile.14 In addition to demonstrating the versatility of protein engineering, these studies have also provided valuable insight into the expected consequences of protein structure modification. For example, it is now recognized that while amino acid substitutions generally lead to only slight structural perturbations, these minor changes in structure can cause significant changes in protein function. Furthermore, it is apparent from several studies with subtilisin that multiple amino acid substitutions may additively affect a particular functional property. Provided with this extensive data base of structure-function relationships in subtilisin, thc cngineering of subtilisin for altered proteolytic activity is now being attempted. Increasing the proteolytic activity of subtilisin could boost the enzyme’s effectiveness as an additive to household laundry detergents. Subtilisin sold for use in laundry detergents accounts for the largest share of the worldwide industrial enzyme market with sales estimated for 1991 at

Selective Protein Degradation by Ligand-Targeted Enzymes: Towards the Creation of Catalytic Antagonists

200 million. Furthermore, the utility of subtilisin for peptide synthesis in aqueous systems can be enhanced by decreasing the enzyme’s proteolytic activity. This would alleviate the problem of low synthesis yields obtained due to proteolysis of the peptide product.

Structural Changes Leading to Increased Enzymatic Activity in an Engineered Variant of Bacillus Lentus Subtilisin

Molecular angler fish: By precisely positioning different binding ligands (L) around the active site “mouth” of a degradative proteinase enzyme, target proteins (TP) can be plucked from solution, locked in position adjacent to the catalytic triad “jaws”, and in this way readily and specifically degraded (see scheme). In this strategy, the appropriate ligand acts as a homing device to confer and enhance selectivity, in the best case by more than 350-fold, in a generic process that exploits the intrinsic, ligand-recognition capabilities of the protein target to trigger its own destruction. The hunting strategy of the deep sea Angler Fish, which uses a lure above its mouth, illustrates this principle.

Engineering Altered Substrate Specificity into Subtilisin BPN

Much of the recent effort of subtilisin protein engineering has centered on the subtilisin from Bacillus lentus. This enzyme has higher alkaline performance than either subtilisin BPN’ from Bacillus amyloliquefaciens or subtilisin Carlsberg from Bacillus licheniformis. While the amino acid sequence of B. lentus subtilisin differs at 106 positions from subtilisin BPN’, including six deleted residues at positions 37a, 58, and 161 to 164, the three-dimensional structures of these subtilisins are very similar and it is possible to draw direct correlations between them.

Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops.

The discovery of restriction enzymes has allowed researchers to undertake far more exact analysis of DNA fragments. It has provided a means of extracting and manipulating specific segments of DNA. Segments of DNA coding for a particular protein can now be incorporated into the genome of a prokaryotic or eukaryotic host in a manner that provides for overexpression of that protein.