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The active site of cross-linked enzyme crystals can be further modified by chemical means, yielding a new type of chemically engineered enzyme. In the past year, the characteristics of cross-linked protein crystals as microporous materials have been studied and used as stationary phases in chromatographic separations.
Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.
A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.
alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate. This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule.
Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, T(eq), arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source ("evolved") temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, T(eq) is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on T(eq) with respect to enzyme temperature/activity effects to be more important than on thermal stability. T(eq) is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution.
Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)
To explore new approaches to enzyme engineering, intra-domain chimeras of two aminoacyl-tRNA synthetases were constructed. Connections were made within the nucleotide folds of these enzymes at sites earlier shown either to be dispensable for activity or able to accommodate oligopeptide insertions. (R.M. Starzyk, T.A. Webster and P. Schimmel, Science 237, 1614 (1987); R.M. Starzyk, J.J. Burbaum and P. Schimmel, Biochemistry, in press). Based on the known structure of one synthetase and structural modeling of the other, the locations of the connection sites allow the possibility of functional "compound" ATP and tRNA binding sites. Of five chimeric genes which were constructed, three direct synthesis of polypeptides that accumulate in vivo. These stable hybrids provide prototypes to which mutagenesis procedures may be applied to produce enzymatically active chimeric synthetases.
In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151alphaF and Q50betaN, showed two- to threefold-increased catalytic efficiency (k(cat)/K(m)) compared to wild-type CA130. Their K(m) values were decreased by ca. 50%, and the k(cat) values increased to 14.4 and 16.9 s(-1), respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121betaA and K198betaA had a 30 to 58% longer half-life than wild-type CA130, and K198betaA and D286betaA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50betaN/K198betaA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.
Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
Investigations in chemical biology have focused on the synthesis of custom-designed proteins with site-specific incorporation of novel amino acids. Their success requires stable production of misacylated tRNAs. Utilization of aminoacyl-tRNA synthetases has been hindered because of enzyme molecular recognition mechanisms that ensure high fidelity of protein synthesis. Leucyl-tRNA synthetase naturally misaminoacylates chemically diverse standard and nonstandard amino acids, but contains a separate amino acid editing active site to hydrolyze incorrectly mischarged tRNAs. In this work, a rational mutagenesis design to block enzyme editing is described and involves substitution of bulky amino acids into the amino acid binding pocket of the hydrolytic active site. These engineered enzymes stably misacylated isoleucine to tRNALeu. The use of these mutant leucyl-tRNA synthetases has the potential to produce pools of mischarged tRNAs that are linked to nonstandard amino acids for in vitro translation. In addition, since many of the leucyl-tRNA synthetases do not interact with or rely upon the tRNA anticodon for identity, these enzymes may offer an excellent scaffold for the development of orthogonal tRNA synthetase/tRNA pairs that can potentially be used to customize protein synthesis.
AIM: Fabry disease is an X-linked inborn error of glycosphingolipid metabolism due to the deficient activity of alpha-galactosidase A, a lysosomal enzyme. It is a multisystem disorder characterized by progressive renal insufficiency, with added morbidity from cardio- and cerebrovascular involvement. The recent availability of genetically engineered enzyme offers an effective targeted treatment approach, but also emphasizes the need for surrogate markers to delineate organ damage and monitor the efficacy of enzyme replacement therapy (ERT). METHODS: Multiple endothelial factors and plasma homocysteine concentrations were investigated in 12 consecutive hemizygous males with classic Fabry disease and 15 controls as part of an exhaustive baseline evaluation prior to ERT. RESULTS: Compared with the controls, plasma concentrations of homocysteine were significantly (p < 0.01) higher in patients with Fabry disease in the absence of chronic renal failure or vitamin deficiency. Plasma concentrations of vascular cell adhesion molecule-1 were also significantly (p < 0.05) higher in the patients, and there was a trend for decreased endothelin-1 levels. No difference was found in serum intercellular adhesion molecule-1, plasma P-selectin, serum E-selectin and plasma thrombomodulin between the patients and controls. CONCLUSIONS: The results do not reveal measurable evidence for endothelial and leukocyte activation that could reliably serve as surrogate markers for routine monitoring of the efficacy of ERT in patients with Fabry disease. While the exact origin and clinical significance of hyperhomocysteinaemia in Fabry disease remains to be studied in a larger cohort of patients carefully monitored for their concurrent medications, especially carbamazepine, we suggest that patients may benefit from folic acid or multivitamin therapy to treat this additional vascular risk factor, when present.
Enzyme engineering is a fast-growing field in the pharmaceutical and food markets. For those applications, various substrates have been examined to immobilize and stabilize enzymes. In this report, we examined peptide nanotubes as supports for enzymes. When a model enzyme, Candida rugosa lipase, was encapsulated in peptide nanotubes, the catalytic activity of nanotube-bound lipases was increased 33% as compared to free-standing lipases at room temperature. At an elevated temperature, 65 degrees C, the activity of lipases inside the nanotubes was 70% higher than free-standing lipases. The activity enhancement of lipases in the peptide nanotubes is likely induced by the conformation change of lipases to the open form (the enzymatically active structure) as lipases are adsorbed on the inner surfaces of peptide nanotubes.
Complete design of the optimum immobilized biocatalyst seems to still be a matter of the future. To be successful, it would require numerical determination of all significant parameters at each enzyme engineering phase, that is at the design of the carriers, immobilized biocatalysts and immobilized reactors. Future research trends should follow this strategy. For processing, cellulosic materials have been considered carriers that fulfill requests to an example model: they represent a unique family of carriers that cover a broad variety of physical and chemical properties, immobilizing techniques, and immobilized reactors as well. The reason for writing this review article was to test the reliability of such a processing and subsequently, to confront theoretical considerations with practical applications of biocatalysts immobilized on cellulose materials.
A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.
The alpha-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis alpha-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.
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Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity. The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain. Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region. As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase. These sites include ones that are flanked by sequences that are conserved between the proteins from E. coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability. We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme. Thus, such an approach may provide for a variety of novel applications.
Recombinant DNA technology, hybridoma technology, enzyme engineering, and protein engineering are all parts of the new biotechnology, which is having a major effect on research in health care, diagnostics, and agriculture, and promises to make inroads in the practices of other industries such as petroleum, mining, foods, and chemicals.