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PubMed1993

Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui.

The gene coding for the enzyme malate dehydrogenase (MDH) of the extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced. The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da. The deduced amino acid sequence of the enzyme was found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs. The structural gene was cloned in the Escherichia coli expression vector pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl. The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria. These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme. Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln. This modification produced an enzyme that has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH). The mutation also caused a modification in the relative activities of the enzyme at different salt concentrations. The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.

Amino Acid Sequence↗
PubMed1969

An immunological approach to the study of evolution of trypsins.

Antibodies were prepared in rabbits against trypsins obtained from four different species-namely bovine, porcine, spiny Pacific dogfish, and starfish (Evasterias trochelii). Each of the antisera, or the immunoglobulin G fraction thereof, was tested for its capacity to react with each of the four enzymes. The immunological reaction was assessed by three different techniques-precipitin reaction, antigen binding capacity, and inhibitory effect of the antibodies on the proteolytic activity of the enzymes. In each case, the homologous enzyme gave the strongest reaction with its antiserum but the heterologous enzymes were also capable of reacting to an appreciable extent. The cross-precipitation was the least sensitive method, and gave relatively low values, whereas cross-inhibition and cross-binding of radioactively labeled antigens indicated high extent of cross-reaction between the various trypsins. The relative capacity of interaction of the four enzymes with the four antibodies could be related to the order in which these enzymes developed during evolution. Thus the "order" of similarity was bovine > porcine > dogfish > starfish.

Animals↗
PubMed1989

The third cellulase of alkalophilic Bacillus sp. strain N-4: evolutionary relationships within the cel gene family.

The third cellulase gene (celC) of Bacillus sp. strain N-4 was cloned in plasmid pBR322 and was located within a 5.5-kb HindIII fragment. The cellulase encoded by this fragment had an Mr of about 100,000 and showed optimum activity around pH 9. These properties were different from those of the enzymes encoded by the celA and celB genes of the same organism. The amino acid sequence deduced from the nucleotide sequence was found to be highly homologous to the CEL-F enzyme from Bacillus sp. strain No. 1139 [Fukumori et al., J. Gen. Microbiol. 132 (1986) 2329-2335]. An evolutionary relationship observed among the four cellulases of alkalophilic Bacillus strains and that of Bacillus subtilis endoglucanase suggested that ancestral genes for alkaline and neutral cellulases diverged early in the evolution of these enzymes.

Amino Acid Sequence↗
PubMed2001

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes.

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.

Alkanes↗
PubMed2004

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution.

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.

Alanine↗
PubMed2005

Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.

Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.

Aspergillus niger↗
PubMed1993

Analysis of cyclic nucleotide-related enzymes by continuous-flow fast-atom bombardment mass spectrometry.

Continuous-flow fast-atom bombardment mass spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.

Animals↗
PubMed2003

4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression.

The enzyme 4-coumarate:CoA ligase (4CL) activates cinnamic acid and its hydroxylated derivatives by forming the corresponding CoA thioesters. These serve as substrates for biosynthesis of phenylpropanoid-derived end-products that are important determinants of fruit quality in raspberry (Rubus idaeus L.). In higher plants, 4CL is typically encoded by a gene family. To investigate the participation of distinct 4CL genes in the process of fruit ripening, we have characterized this gene family in raspberry. By complementing a PCR-based homology search with low-stringency cDNA library screening, we have isolated three classes of raspberry 4CL cDNAs (Ri4CL1, Ri4CL2, and Ri4CL3). Phylogenetic analysis places the three raspberry 4CL gene family members into two distinct groups, a pattern consistent with an ancient divergence from an ancestral progenitor. Quantitative RT-PCR assay reveals a differential pattern of transcription of each of the three genes in various organs, as well as distinct temporal patterns of expression during flower and fruit development. The regulatory elements thus appear to have evolved independently of the genes themselves. Based on phylogenetic classification, expression patterns and recombinant protein activities the different Ri4CL genes are likely to participate in different biosynthetic pathways leading to the various phenylpropanoid-derived metabolites that help create flavor and color in raspberry fruit.

Cloning, Molecular↗
PubMed2004

GDSL family of serine esterases/lipases.

GDSL esterases and lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of important ester compounds of pharmaceutical, food, biochemical, and biological interests. This new subclass of lipolytic enzymes possesses a distinct GDSL sequence motif different from the GxSxG motif found in many lipases. Unlike the common lipases, GDSL enzymes do not have the so called nucleophile elbow. Studies show that GDSL hydrolases have a flexible active site that appears to change conformation with the presence and binding of the different substrates, much like the induced fit mechanism proposed by Koshland. Some of the GDSL enzymes have thioesterase, protease, arylesterase, and lysophospholipase activity, yet they appear to be the same protein with similar molecular weight ( approximately 22-60 kDa for most esterases), although some have multiple glycosylation sites with higher apparent molecular weight. GDSL enzymes have five consensus sequence (I-V) and four invariant important catalytic residues Ser, Gly, Asn, and His in blocks I, II, III, and V, respectively. The oxyanion structure led to a new designation of these enzymes as SGNH-hydrolase superfamily or subfamily. Phylogenetic analysis revealed that block IIA which belonged to the SGNH-hydrolases was found only in clade I. Therefore, this family of hydrolases represents a new example of convergent evolution of lipolytic enzymes. These enzymes have little sequence homology to true lipases. Another important differentiating feature of GDSL subfamily of lipolytic enzymes is that the serine-containing motif is closer to the N-terminus unlike other lipases where the GxSxG motif is near the center. Since the first classification of these subclass or subfamily of lipases as GDSL(S) hydrolase, progress has been made in determining the consensus sequence, crystal structure, active site and oxyanion residues, secondary structure, mechanism of catalysis, and understanding the conformational changes. Nevertheless, much still needs to be done to gain better understanding of in vivo biological function, 3-D structure, how this group of enzymes evolved to utilize many different substrates, and the mechanism of reactions. Protein engineering is needed to improve the substrate specificity, enantioselectivity, specific activity, thermostability, and heterologous expression in other hosts (especially food grade microorganisms) leading to eventual large scale production and applications. We hope that this review will rekindle interest among researchers and the industry to study and find uses for these unique enzymes.

Amino Acid Sequence↗
PubMed2003

Prediction of the structure and function of AstA and AstB, the first two enzymes of the arginine succinyltransferase pathway of arginine catabolism.

Arginine succinyltransferase and succinylarginine dihydrolase catalyze the first two steps of arginine catabolism by the arginine succinyltransferase pathway. This route is the only major arginine catabolic pathway in Escherichia coli including its pathogenic strains O157 and CFT073. We have used fold recognition tools and identified novel homologies between each of these two enzymes and proteins of known three-dimensional structure: arginine succinyltransferase belongs to the acyl-CoA N-acyltransferase superfamily and succinylarginine dihydrolase belongs to the amidinotransferase superfamily. These findings shed light on the structures, catalytic mechanisms and evolution of diverse enzymes involved in arginine catabolism.

Acyltransferases↗
PubMed1991

Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.

Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.

Base Sequence↗
PubMed2000

From no-confidence to nitric oxide acknowledgement: a story of bacterial nitric-oxide reductase.

The review briefly summarizes current knowledge of the bacterial nitric-oxide reductase (NOR). This membrane enzyme consists of two subunits, the smaller one contains haem C and the larger one two haems B and nonhaem iron. The protein sequence and structure of metal centres demonstrate the relationship of NOR to the family of terminal oxidases. The binuclear Fe-Fe reaction centre, consisting of antiferromagnetically coupled haem B and nonhaem iron, is analogous to Fe-Cu centre of terminal oxidases. The data on the structure and function of NOR and terminal oxidases suggest that all these enzymes are closely evolutionally related. The catalytic properties are determined most of all by the relatively high toxicity of nitric oxide as a substrate and the resulting strong need to maintain its concentration at nanomolar levels. A kinetic model of the action of the enzyme comprises substrate inhibition. NOR does not conserve the free energy of nitric oxide reduction because it does not work as a proton pump and, moreover, the protons coming into the reaction are taken from periplasm, i.e. they do not cross the membrane.

Bacteria↗
PubMed2003

Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin.

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.

Animals↗
PubMed1985

Limits of adaptation: the evolution of selective neutrality.

Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form. The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness. Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number. At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral. We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection. Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli. Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways. We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations. The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.

Adaptation, Physiological↗
PubMed1978

Structure of pyruvate kinase and similarities with other enzymes: possible implications for protein taxonomy and evolution.

The structure determination of pyruvate kinase shows that each subunit of the tetrameric molecule consists of three domains. The largest of these domains has a remarkable similarity to the structure of triosephosphate isomerase. Another domain shows similarities to many other nucleotide binding proteins. We discuss these similarities and their implications for current arguments on protein taxonomy and evolution.

Animals↗
PubMed1977

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Adenosine Deaminase↗