Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development

As isoprenoid biosynthetic pathway has gained importance since last few years, key enzymes of this pathway have been characterized and their functional roles in the cell metabolism have been explored using molecular biology approaches. A key enzyme in this pathway is farnesyl pyrophosphate (EC 2.5.1.10) synthase (FPPS) which supplies precursors for the biosynthesis of essential isoprenoids like carotenoids, withanolides, ubiquinones, dolichols, sterols, among others and also helps in farnesylation and geranylation of proteins. It is a chain elongation enzyme which catalyzes head to tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl pyrophosphate (FPP). Recent studies have validated FPPS as a molecular target of bisphosphonates for drug development against tumors as well as human pathogens. The present paper synthesizes the information on characterization, structural and functional relationships, evolution, localization as well as advances on FPPS enzyme as a target for drug development.     

Circular Permutations in the Molecular Evolution of DNA Methyltransferases

Circular permutations of genes during molecular evolution often are regarded as elusive, although a simple model can explain these rearrangements. The model assumes that first a gene duplication of the precursor gene occurs in such a way that both genes become fused in frame, leading to a tandem protein. After generation of a new start codon within the 5′ part of the tandem gene and a stop at an equivalent position in the 3′ part of the gene, a protein is encoded that represents a perfect circular permutation of the precursor gene product. The model is illustrated here by the molecular evolution of adenine-N⁶ DNA methyltransferases. β- and γ-type enzymes of this family can be interconverted by a single circular permutation event. Interestingly, tandem proteins, proposed as evolutionary intermediates during circular permutation, can be directly observed in the case of adenine methyltransferases, because some enzymes belonging to type IIS, like the FokI methyltransferase, are built up by two fused enzymes, both of which are active independently of each other. The mechanism for circular permutation illustrated here is very easy and applicable to every protein. Thus, circular permutation can be regarded as a normal process in molecular evolution and a changed order of conserved amino acid motifs should not be interpreted to argue against divergent evolution. Received: 17 November 1998 / Accepted: 19 February 1999     

The Effect of Branch Length Variation on the Selection of Models of Molecular Evolution

Models of sequence evolution play an important role in molecular evolutionary studies. The use of inappropriate models of evolution may bias the results of the analysis and lead to erroneous conclusions. Several procedures for selecting the best-fit model of evolution for the data at hand have been proposed, like the likelihood ratio test (LRT) and the Akaike (AIC) and Bayesian (BIC) information criteria. The relative performance of these model-selecting algorithms has not yet been studied under a range of different model trees. In this study, the influence of branch length variation upon model selection is characterized. This is done by simulating sequence alignments under a known model of nucleotide substitution, and recording how often this true model is recovered by different model-fitting strategies. Results of this study agree with previous simulations and suggest that model selection is reasonably accurate. However, different model selection methods showed distinct levels of accuracy. Some LRT approaches showed better performance than the AIC or BIC information criteria. Within the LRTs, model selection is affected by the complexity of the initial model selected for the comparisons, and only slightly by the order in which different parameters are added to the model. A specific hierarchy of LRTs, which starts from a simple model of evolution, performed overall better than other possible LRT hierarchies, or than the AIC or BIC. Received: 2 October 2000 / Accepted: 4 January 2001     

Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.     

Tyrosine metabolic enzymes from insects and mammals：A comparative perspective

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess finetuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modem genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precur sor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mam malian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.     

A new intrinsic thermal parameter for enzymes reveals true temperature optima

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.     

Directing the evolution of Rubisco and Rubisco activase: first impressions of a new tool for photosynthesis research

During the last decade the practice of laboratory-directed protein evolution has become firmly established as a versatile tool in biochemical research by enabling molecular evolution toward desirable phenotypes or detection of novel structure-function interactions. Applications of this technique in the field of photosynthesis research are still in their infancy, but recently first steps have been reported in the directed evolution of the CO(2)-fixing enzyme Rubisco and its helper protein Rubisco activase. Here we summarize directed protein evolution strategies and review the progressive advances that have been made to develop and apply suitable selection systems for screening mutant forms of these enzymes that improve the fitness of the host organism. The goal of increasing photosynthetic efficiency of plants by improving the kinetics of Rubisco has been a long-term goal scoring modest successes. We discuss how directed evolution methodologies may one day be able to circumvent the problems encountered during this venture.     

Identification and characterisation of five novel miniature inverted-repeat transposable elements (MITEs) in amphioxus (Branchiostoma floridae)

As the sister group to vertebrates, amphioxus is consistently used as a model of genome evolution for understanding the invertebrate/vertebrate transition. The amphioxus genome has not undergone massive duplications like those in the vertebrates or disruptive rearrangements like in the genome of Ciona, a urochordate, making it an ideal evolutionary model. Transposable elements have been linked to many genomic evolutionary changes including increased genome size, modified gene expression, massive gene rearrangements, and possibly intron evolution. Despite their importance in genome evolution, few previous examples of transposable elements have been identified in amphioxus. We report five novel Miniature Inverted-repeat Transposable Elements (MITEs) identified by an analysis of amphioxus DNA sequence, which we have named LanceleTn-1, LanceleTn-2, LanceleTn-3a, LanceleTn-3b and LanceleTn-4. Several of the LanceleTn elements were identified in the amphioxus ParaHox cluster, and we suggest these have had important implications for the evolution of this highly conserved gene cluster. The estimated high copy numbers of these elements implies that MITEs are probably the most abundant type of mobile element in amphioxus, and are thus likely to have been of fundamental importance in shaping the evolution of the amphioxus genome.     

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 (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.     

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.     

Is the microbial tree of life verificationist?

The field of microbial phylogenetics has questioned the feasibility of using a tree‐like structure to the describe microbial evolution. This debate centres on two main points. First, because microorganisms are able to transfer genes from one to another in zero generations (horizontal gene transfer, or HGT), the use of molecular characters to perform phylogenetic analyses will yield an erroneous topology and HGT clearly makes the evolution of microorganisms non tree‐like. Second, the use of concatenated gene sequences in a total evidence approach to phylogenetic systematics is a verificationist endeavour, the aim of which is to bolster support. However, the goal of the total evidence approach to phylogenetic research is based in the idea of increasing explanatory power over background knowledge through test and corroboration, rather than to bolster support for nodes in a tree. In this context, the testing of phylogenetic data is a falsificationist endeavour that includes the possibility of not rejecting the null hypothesis that there is no tree‐like structure in molecular phylogenetic data. We discuss several tests that aim to test rigorously the hypothesis that a tree of life exists for microorganisms. We also discuss the philosophical ramifications of background knowledge and corroboration in microbial studies that need to be considered when suggesting that HGT confounds the tree of life. © The Willi Hennig Society 2009.     

Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock?

Summary We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts. Offprint requests to: A. Moya     

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.     

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature’s biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

     

Microsatellite length differences between humans and chimpanzees at autosomal Loci are not found at equivalent haploid Y chromosomal Loci

When homologous microsatellites are compared between species, significant differences in mean length are often noted. A dominant cause of these length differences is ascertainment bias due to selection for maximum repeat number and repeat purity when the markers are being developed. However, even after ascertainment bias has been allowed for through reciprocal comparisons, significant length differences remain, suggesting that the average microsatellite mutation rate differs between species. Two classes of mechanism have been proposed: rapid evolution of enzymes involved in the generation and repair of slippage products (enzyme evolution model) and heterozygote instability, whereby interchromosomal events at heterozygous sites offer extra opportunities for mutations to occur (heterozygote instability model). To examine which of these hypotheses is most likely, we compared ascertainment bias and species length differences between humans and chimpanzees in autosomal and Y chromosomal microsatellites. We find that levels of ascertainment bias are indistinguishable, but that interspecies length differences are significantly greater for autosomal loci compared with haploid Y chromosomal loci. Such a pattern is consistent with predictions from the heterozygote instability model and is not expected under models of microsatellite evolution that do not include interchromosomal events such as the enzyme evolution model.     

溶菌酶的研究进展

溶菌酶普遍存在于动物、植物和微生物中。溶菌酶是一种小分子碱性蛋白,长期以来一直被作为一种“模型”体系．用于研究蛋白质的空间构象、酶动力学及其与分子进化、分子免疫间的关系。介绍了溶菌酶的来源、结构、性质、作用机制。并对近年来其在食品工业、医学和酶工程中的应用进行了综述;分析了溶菌酶应用中存在的主要问题,并对其应用前景进行了展望。     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

The nearly neutral and selection theories of molecular evolution under the fisher geometrical framework: substitution rate, population size, and complexity

The general theories of molecular evolution depend on relatively arbitrary assumptions about the relative distribution and rate of advantageous, deleterious, neutral, and nearly neutral mutations. The Fisher geometrical model (FGM) has been used to make distributions of mutations biologically interpretable. We explored an FGM-based molecular model to represent molecular evolutionary processes typically studied by nearly neutral and selection models, but in which distributions and relative rates of mutations with different selection coefficients are a consequence of biologically interpretable parameters, such as the average size of the phenotypic effect of mutations and the number of traits (complexity) of organisms. A variant of the FGM-based model that we called the static regime (SR) represents evolution as a nearly neutral process in which substitution rates are determined by a dynamic substitution process in which the population's phenotype remains around a suboptimum equilibrium fitness produced by a balance between slightly deleterious and slightly advantageous compensatory substitutions. As in previous nearly neutral models, the SR predicts a negative relationship between molecular evolutionary rate and population size; however, SR does not have the unrealistic properties of previous nearly neutral models such as the narrow window of selection strengths in which they work. In addition, the SR suggests that compensatory mutations cannot explain the high rate of fixations driven by positive selection currently found in DNA sequences, contrary to what has been previously suggested. We also developed a generalization of SR in which the optimum phenotype can change stochastically due to environmental or physiological shifts, which we called the variable regime (VR). VR models evolution as an interplay between adaptive processes and nearly neutral steady-state processes. When strong environmental fluctuations are incorporated, the process becomes a selection model in which evolutionary rate does not depend on population size, but is critically dependent on the complexity of organisms and mutation size. For SR as well as VR we found that key parameters of molecular evolution are linked by biological factors, and we showed that they cannot be fixed independently by arbitrary criteria, as has usually been assumed in previous molecular evolutionary models.     

Evolution of enzymes in metabolism: a network perspective

Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.