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PubMedDate not supplied

Designing enzymes for use in organic solvents.

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.

Binding Sites↗
PubMed1997

[Structures and functions of ribonucleases].

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.

Amino Acid Sequence↗
PubMed2004

Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

Acetamides↗
PubMed1996

Construction of a P450c27 fusion enzyme: a useful tool for analysis of vitamin D3 25-hydroxylase activity.

Liver mitochondrial P450c27, encoded by the CYP27 gene, can catalyse the 25-hydroxylation of vitamin D3 and the 27-hydroxylation of sterols. To facilitate the study of this enzyme in cell culture systems, we engineered a fusion protein consisting of P450c27 coupled to its electron-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side-chain) hydroxylation of 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3). When incubated with 1 alpha-OH-D3, COS-1 cells transfected with a vector expressing the fusion protein produced 1 alpha,25-(OH)2D2 and 1 alpha,27-(OH)2D3 about four times more efficiently than did cells transfected with three individual components of the fusion. When incubated with the natural substrate, vitamin D3, the efficiency of hydroxylation was lower than that for 1 alpha-OH-D3 but still 1.7-fold higher for the fusion protein than for its individual components. The fusion protein was also able to reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27-ferredoxin reductase-ferredoxin fusion construct represents a valuable tool for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.

Animals↗
PubMed2005

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains.

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Amino Acid Sequence↗
PubMed1994

Drosophila lebanonensis ADH: analysis of recombinant wild-type enzyme and site-directed mutants. The effect of restoring the consensus sequence in two positions.

Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD(+)-binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus amino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13Gly and Asn56Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.

Alcohol Dehydrogenase↗
PubMed1989

Isolation, characterization and manipulation of cellulase genes.

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.

Journal Article↗
PubMedDate not supplied

Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

Aryldialkylphosphatase↗
PubMed1994

Bacteriophage T4 gene 55.9 encodes an activity required for anaerobic ribonucleotide reduction.

Bacteriophage T4 contains a phage-encoded anaerobic ribonucleoside triphosphate reductase, nrdD, previously named sunY. An open reading frame, 55.9, that resides downstream of the phage reductase was observed to have amino acid sequence similarity with the E. coli pyruvate formate-lyase (Pfl) activating enzyme. A stop codon was engineered into the cloned 55.9 gene and then recombined back into the phage genome. Phage-infected extracts that lack a functional 55.9 product have a 6-fold reduction in anaerobic ribonucleotide reductase activity and are unable to activate overexpressed T4 NrdD. Restoration of reductase activity was possible when 55.9- and nrdD- T4-infected Escherichia coli extracts were conjointly assayed. Comparing the anaerobic burst size of 55.9- infections to that of the parental phage indicates that anaerobic de novo synthesis of deoxyribonucleotides is nearly abolished in phage lacking the 55.9 product. We propose that T4 55.9 encodes an enzyme that activates T4 NrdD by generating a glycyl radical in the phage-encoded reductase. The homology between the Pfl activating enzyme and T4 55.9 product (in this communication renamed NrdG) in function as well as amino acid sequence is presumably a remnant of an ancient heritage between Pfl and the anaerobic ribonucleotide reductases.

Acetyltransferases↗
PubMedDate not supplied

Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress.

Hemoglobins in unicellular organisms, like the one here in the bacterium Vitreoscilla, have greater chemical reactivity than their homologues in multicellular organisms. They can catalyze redox reactions and may protect cells against oxidative stress. The ability of Vitreoscilla hemoglobin to complement deficiencies of terminal cytochrome oxidases in Escherichia coli also suggests that this hemoglobin can receive electrons during respiration. In this study, a recombinant strain of Enterobacter aerogenes engineered to produce the Vitreoscilla Hb was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress produced by exogenously applied hydrogen peroxide was characterized in terms of cell growth, survival and the activities of two key antioxidant enzymes (catalase and superoxide dismutase). The influence of the physiological state of the cells and different media upon these culture dynamics was determined. Results showed that the hemoglobin-expressing strain is quite distinct in terms of growth/survival properties and activity of antioxidant enzymes from that of non-hemoglobin counterparts.

Bacterial Proteins↗
PubMed1997

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo.

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

Cloning, Molecular↗
PubMed2002

Milestones in directed enzyme evolution.

Directed evolution has now been used for over two decades as an alternative to rational design for protein engineering. Protein function, however, is complex, and modifying enzyme activity is a tall order. We can now improve existing enzyme activity, change enzyme selectivity and evolve function de novo using directed evolution. Although directed evolution is now used routinely to improve existing enzyme activity, there are still only a handful of examples where substrate selectivity has been modified sufficiently for practical application, and the de novo evolution of function largely eludes us.

Catalysis↗
PubMed2001

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.

Amino Acid Sequence↗
PubMed1992

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.

Animals↗
PubMed2002

Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain.

Matrix metalloproteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several central nervous system disorders. Increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer's disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis. This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity. To allow us to evaluate the effect of increased levels of active MMP-9 in the central nervous system, mutant forms of the enzyme were designed to autocatalytically remove the pro domain, yielding active enzyme. This was accomplished by modifying residues in the cysteine switch autoinhibitor region of the propeptide. Stable cell lines and transgenic mice that express G100L and D103N autoactive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.

Amino Acid Sequence↗
PubMed1993

Engineered yeast cells as model to study coupling between human xenobiotic metabolizing enzymes. Simulation of the two first steps of benzo[a]pyrene activation.

Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH-cytochrome P450 reductase (P450 reductase). A styrene-oxide-hydrolase activity (2 nmol.min-1.mg microsomal protein-1) and a 7-ethoxyresorufin-O-deethylase activity (320 pmol.min-1.mg microsomal protein-1) characteristic respectively of microsomal epoxide hydrolase and P450 1A1 were detected. The conversion of benzo[a]pyrene (B[a]P) to B[a]P-7,8-dihydrodiol both in microsomal preparations and in growing yeast cells was observed, demonstrating an efficient coupling between the two human enzymes. Kinetic analysis indicated that the B[a]P-7,8-oxide produced by the P450-1A1-dependent reaction does not accumulate before hydrolysis by microsomal epoxide hydrolase. This system was also used as a control to evaluate the coupling efficiency of a mixture of microsomes or of yeast cells containing separately the individual enzymes (i.e., human P450 1A1 and microsomal epoxide hydrolase). B[a]P-7,8-oxide was well converted to the corresponding dihydrodiol with a mixture of microsomes. In contrast, when the same experiment was repeated with a mixture of cells expressing independently the two activities, dihydrodiol formation was not observed. Coexpression of human phase I and phase II enzymes in a single yeast cell and microsome mixture thus appear to be complementary tools for the simulation of human-drug-metabolism or carcinogen-metabolism pathways.

Base Sequence↗
PubMed2002

Yeast cytochrome c peroxidase: mechanistic studies via protein engineering.

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c. It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity. Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen-oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The catalytic mechanism involves complex formation between cytochrome c and CcP. The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein-protein recognition, complex formation, and intracomplex electron transfer processes.

Catalytic Domain↗