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PubMed1996

Engineering enzymes for stability.

There have been many recent developments in elaborating the approaches for stabilizing enzymes by stabilizing the folding state, destabilizing the unfolded state and altering the kinetics of unfolding. However, these represent a series of rules of thumb rather than the reliable principles that would be expected of 'engineering'. Stability is taken to include thermodynamic stability as measured by reversible denaturation, and kinetic stability as measured by the unfolding rate for enzymes that are subject to irreversible denaturation. As such, the factors that affect stability of the folded state versus the unfolded state, as well as factors affecting rates of folding and unfolding, all play a role in maintaining stability. Recent studies on the role of kinetics and the effect that site-specific substitutions have on transition-state free energies add to our understanding of the factors that determine whether or not a particular substitution will result in the measurable stabilization of a protein.

Enzyme Stability↗
PubMed1990

Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.

Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.

Amino Acid Sequence↗
PubMed2001

Investigating and Engineering Enzymes by Genetic Selection.

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.

Journal Article↗
PubMed2007

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.

2-Naphthylamine↗
PubMed1993

Engineering enzymes for clinical diagnosis.

The enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3.) is a major limiting factor in the enzymatic creatinine determination because of its comparatively poor catalytic activity and stability in the native form. The gene from Pseudomonas putida coding for creatinase was cloned and used for overexpression of the protein in E coli and Pseudomonas. In addition, it was possible by means of 'random' mutagenesis in vivo and subsequent screening using an activity plate assay to isolate creatinase derivatives that are more stable towards detergents and elevated temperature under test kit conditions. This example shows that enzymes can be optimized for use in given assay conditions by mutagenesis of cloned DNA and suitable screening methods.

Cloning, Molecular↗
PubMed1988

Biocatalysis made to order.

Recombinant DNA technology is now being explored to engineer enzyme molecules. It has many far-reaching applications in biocatalytic processes of enzyme engineering. The facts have pursued certain important industrial, biomedical, and environmental problems. These current excitements are mainly focused on the basis of gene cloning and in vitro mutagenesis for overproduction and redesigning of enzymes, as well as their probable implications in industry, antibiotic research, and waste degradation.

Catalysis↗
PubMed2006

The thermal behaviour of enzyme activity: implications for biotechnology.

The way that enzymes respond to temperature is fundamental to many areas of biotechnology. This has long been explained in terms of enzyme stability and catalytic activation energy, but recent observations of enzyme behaviour suggest that this picture is incomplete. We have developed and experimentally validated a new model to describe the effect of temperature on enzymes; this model incorporates additional fundamental parameters that enable a complete description of the effects of temperature on enzyme activity. In this article, we consider the biotechnological implications of this model in the areas of enzyme engineering, enzyme reactor operation and the selection and/or screening of useful enzymes from the environment.

Bioreactors↗
PubMed2002

Oxidative biotransformations using oxygenases.

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.

Biotransformation↗
PubMed1999

Chemical engineering of enzymes: altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin.

The increasing demand for enzymes as highly selective, mild, and environmentally benign catalysts is often limited by the lack of an enzyme with the desired catalytic activity or substrate selectivity and by their instability in biotechnological processes. The previous answers to these problems comprised genetically engineered enzymes and several classes of enzyme mimics. Here we describe the potential of chemical enzyme engineering: native enzymes can be modified by merely chemical means and basic equipment yielding so-called semisynthetic enzymes. Thus, the high substrate selectivity of the enzymatic peptide framework is combined with the catalytic versatility of a synthetic active site. We illustrate the potential of chemically engineered enzymes with the conception of the semisynthetic peroxidase seleno-subtilisin. First, the serine endoprotease subtilisin was crystallized and cross-linked with glutaraldehyde to give cross-linked enzyme crystals which were found to be insoluble in water or organic solvents and highly stable. Second, serine 221 in the active site (Enz-OH) was chemically converted into an oxidized derivative of selenocystein (Enz-SeO2H). As a consequence, the former proteolytic enzyme gained peroxidase activity and catalyzed the selective reduction of hydroperoxides. Due to the identical binding sites of the semisynthetic peroxidase and the protease, the substrate selectivity of seleno-subtilisin was predictable in view of the well-known selectivity of subtilisin.

Binding Sites↗
PubMed1992

Engineered metalloregulation in enzymes.

Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.

Amino Acid Sequence↗