STUDIES OF A MAMMALIAN ENZYME SYSTEM FOR PRODUCING EVOLUTIONARY EVIDENCE ON MAN.
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Animals↗
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Results for “enzyme evolution”
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Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases.
The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (EC 4.1.2.11) is involved in the catabolism of the cyanogenic glycoside dhurrin. We have isolated a cDNA clone comprising about 90% of the COOH terminal sequence of a precursor which encodes both subunit of HNL from Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be the result of post-translational processing. The deduced amino acid sequence of HNL shares significant sequence homology with members of the serine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp. His, and Ser with these enzymes which evolved in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtilisin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linked A and B chains forming a heterotetramer of a molecular weight of 105,000 (carboxypeptidases 120,000). Thus, HNL from sorghum closely resembles to serine carboxypeptidases but differs from all other HNLs described so far. Western blotting experiments revealed cross reaction between carboxypeptidase from wheat and anti SbHNL antisera. Therefore, convergent evolution of HNLs from various ancestoral enzymes is conceivable. Hybridization of SbHNL cDNA to northern blots of total RNAs isolated from various organs of young sorghum seedlings shows the same expression pattern of HNL as found by means of western blotting or enzyme assays. Using PCR and Southern blot analysis, we demonstrated that the gene of SbHNL is free of introns. Further sequence analysis of cDNA clones and genomic DNA revealed a stretch of 23 adenine residues in the 3'-untranslated part of the gene. Both, intronless organisation of the gene and a genomic stretch of oligo A suggests that SbHNL may have evolved by a reverse transcription event.
Aldehyde-Lyases↗
Enantioselective biocatalysis optimized by directed evolution.
Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.
Algorithms↗
In vivo molecular evolution reveals biophysical origins of organismal fitness.
In nature, evolution occurs through the continuous adaptation of a population to its environment. At the molecular level, adaptive changes in protein sequence and expression impact organismal fitness and, consequently, dictate population dynamics. Here, we have used a "weak link" method to favor variations in one gene, allowing adaptation to thermostability to be studied in molecular detail as bacteria were grown continuously for approximately 1500 generations. Surprisingly, only six mutant alleles, representing less than 1% of the possible missense mutations, were observed, suggesting a highly constrained molecular landscape during protein evolution. The changes in organismal fitness were linked directly to incremental increases in enzyme stability and activity maxima and corresponded to the narrow temperature ranges where each mutant enjoyed success within the overall population. Thus, continuous evolution of a single gene permits a quantitative approach that extends from the phenotypes of the microbial populations to their underlying biophysical basis.
Adenylate Kinase↗
Function, structure and evolution of fructose-1,6-bisphosphatase.
The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.
Amino Acid Sequence↗
Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?
Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels.
Animals↗
Genomic organization of the mouse and human genes encoding the ATP sulfurylase/adenosine 5'-phosphosulfate kinase isoform SK2.
Mammalian ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase consists of kinase and sulfurylase domains, and catalyzes two sequential reactions to synthesize the universal sulfate donor, phosphoadenosine phosphosulfate (PAPS). In simpler organisms, the ATP sulfurylase and APS kinase reactions are catalyzed by separate enzymes encoded by two or three genes, suggesting that a fusion of separate genes during the course of evolution generated the bifunctional enzyme. We have characterized the genomic structure of the PAPS synthetase SK2 isoform genes for mouse (MSK2) and human (HSK2) and analyzed the possible fusion region. The MSK2 and HSK2 genes exhibit a common structure of 13 exons, including a 15-nucleotide alternatively spliced exon 8. Enzyme activities of several bacterially expressed exon assemblages showed exons 1-6 encode APS kinase, while exons 6-13 encode ATP sulfurylase. The MSK2 construct without the exon 6-encoded peptide showed no kinase or sulfurylase activity, demonstrating that exon 6 encodes sequences required for both activities. Exon 1 and its 5'-flanking sequence are highly divergent between the two species, and intron 1 of the HSK2 gene contains a region similar to the MSK2 promoter sequence, suggesting that it may be the remnant of a now-superceded regulatory region. The HSK2 promoter contains a GC-rich region, not present in the mouse promoter, and has few transcription factor binding sites in common with MSK2. These differences in the two promoter regions suggest that species-specific mechanisms regulate expression of the SK2 isoform.
Amino Acid Sequence↗
Genetic variation affecting the expression of enzyme-coding genes in Drosophila: an evolutionary perspective.
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Animals↗
Post-translational self-hydroxylation: a probe for oxygen activation mechanisms in non-heme iron enzymes.
Recent years have seen considerable evolution in our understanding of the mechanisms of oxygen activation by non-heme iron enzymes, with high-valent iron-oxo intermediates coming to the forefront as formidably potent oxidants. In the absence of substrate, the generation of vividly colored chromophores deriving from the self-hydroxylation of a nearby aromatic amino acid for a number of these enzymes has afforded an opportunity to discern the conditions under which O2 activation occurs to generate a high-valent iron intermediate, and has provided a basis for a rigorous mechanistic examination of the oxygenation process. Here, we summarize the current evidence for self-hydroxylation processes in both mononuclear non-heme iron enzymes and in mutant forms of ribonucleotide reductase, and place it within the context of our developing understanding of the oxidative transformations accomplished by non-heme iron centers.
Hydroxylation↗
Homology, pathway distance and chromosomal localization of the small molecule metabolism enzymes in Escherichia coli.
Here, we analyse Escherichia coli enzymes involved in small molecule metabolism (SMM). We introduce the concept of pathway distance as a measure of the number of distinct metabolic steps separating two SMM enzymes, and we consider protein homology (as determined by assigning enzymes to structural and sequence families) and gene interval (the number of genes separating two genes on the E. coli chromosome). The relationships between these three contexts (pathway distance, homology and chromosomal localisation) is investigated extensively. We make use of these relationships to suggest possible SMM evolution mechanisms. Homology between enzyme pairs close in the SMM was higher than expected by chance but was still rare. When observed, homologues usually conserved their reaction mechanism and/or co-factor binding rather than shared substrate binding. The correlation between pathway distance and gene intervals was clear. Enzymes catalysing nearby SMM reactions were usually encoded by genes close by on the E. coli chromosome. We found many co-regulated blocks of three to four genes (usually non-homologous) encoding enzymes occurring within four metabolic steps of one another; nearly all of these blocks formed part of known or predicted operons. The "inline reuse" of enzymes (i.e. the use of the same enzyme to catalyse two or more different steps of a metabolic pathway) is also discussed: of these enzymes, four were multifunctional (i.e. catalysed a different reaction in each instance), nine had multiple substrate specificity (i.e. catalysed the same reaction on different substrates in each instance) and one catalysed the same reaction on the same substrate but as part of two different complexes. We also identified 59 sets of isozymic proteins most commonly duplicated to function under different conditions, or with a different preferred substrate or minor substrate. In addition to transcriptional units, isozymes and inline reuse of enzymes provide mechanisms for controlling the SMM network. Our data suggest that several pathway evolution mechanisms may occur in concert, although chemistry-driven duplication/recruitment is favoured. SMM exploits regulatory strategies involving chromosomal location, isozymes and the reuse of enzymes.
Chromosomes, Bacterial↗
Structure of Escherichia coli aminodeoxychorismate synthase: architectural conservation and diversity in chorismate-utilizing enzymes.
Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.
Amino Acid Sequence↗
Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution.
We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.
Amino Acid Motifs↗
Mitochondrial enzyme content in the muscles of high-performance fish: evolution and variation among fiber types.
Muscle mitochondrial content varies widely among fiber types and species. We investigated the origins of variation in the activity of the mitochondrial enzyme citrate synthase (CS), an index of mitochondrial abundance, among fiber types and species of high-performance fish (tunas and billfishes). CS activities varied up to 30-fold among muscles: lowest in billfish white muscle and highest in billfish heater organ. Among species, CS activities of red, white, and cardiac muscles of three tuna species were twofold greater than the homologous muscles of two billfish species. Because comparisons of CS amino acid sequences deduced from a combination of PCR methods argue against clade-specific differences in catalytic properties, CS activity reflects CS content among these five species. To assess the bases of these differences in CS activity, we looked at the relationship between CS activity (U/g muscle), nuclear content (DNA/g muscle), and CS transcript levels (CS mRNA/g RNA). Muscle CS activity differed by 10- to 30-fold when expressed per gram of muscle but only threefold when expressed per milligram of DNA. Thus it is nuclear DNA content, not fiber-type differences, in CS gene expression that may be the main determinant of CS activity in muscle. Conversely, evolutionary (tunas vs. billfishes) differences in CS arise from differences in posttranscriptional regulation, based on relationships between CS enzyme levels and CS mRNA assessed by quantitative competitive RT-PCR. These data argue that fiber-type differences can arise without major differences in fiber-type-specific regulation of the CS gene, whereas evolutionary differences may be largely due to posttranscriptional regulation.
Amino Acid Sequence↗
A classification of glycosyl hydrolases based on amino acid sequence similarities.
The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.
Amino Acid Sequence↗
[Current researches on molecular enzyme engineering].
The developments of recombinant DNA technology and structural biology make it possible to modify enzyme in molecular level. Scientists show growing interests in the evolution or functional fusion of enzymes. Recent advances and applications of the molecular enzyme engineering are reviewed and discussed in this article.
Enzymes↗
The population genetics of alleles affecting enzyme activity.
It is possible to predict the population genetics of allozymes by assuming that fitness is proportional to flux through a biochemical pathway. The model presented here extends previous work by incorporating two additional features of biological realism. Firstly, that more than one biochemical route may exist between any two metabolites. The major routes have been identified as the classical biochemical pathways but in the event of a mutation blocking a major route, minor routes become significant. These minor routes are named "bypass fluxes" and have profound effects on the population genetics of allozymes. Secondly, recent work has suggested that a metabolic cost is associated with enzyme synthesis; this will constitute an additional selective pressure on alleles which affect the amount of enzyme synthesized. The model generates a fitness curve which predicts the fitness associated with any level of enzyme activity. It can utilize data on null or near-null, structural or regulatory, mutations in the presence or absence of bypass fluxes. When data from natural populations of Drosophila are investigated, it is concluded that selection pressures acting on enzyme variants may be much higher than previously thought.
Animals↗
The development of enzyme catalytic efficiency: an experimental approach.
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Binding Sites↗
RNase P from a photosynthetic organelle contains an RNA homologous to the cyanobacterial counterpart.
RNase P is a ribonucleoprotein enzyme in all organisms and organelles investigated so far, with the exception of chloroplasts where no enzyme-associated RNA has been detected to date. As an approach to answer the question whether an RNA component is present in RNase P from photosynthetic organelles, we have used a phylogenetically oriented strategy and searched for RNase P RNA in a postulated intermediate in plastid evolution, the cyanelle of Cyanophora paradoxa. We have detected a 351 nucleotide long RNA similar to cyanobacterial RNase P RNAs, with a proposed secondary structure that closely resembles a bacterial consensus. The RNA is encoded on the cyanelle genome and copurifies with enzyme activity. The RNA is not catalytically active by itself, but the activity of the cyanelle RNase P holoenzyme is destroyed by nuclease treatment, indicating an essential role of the RNA. Hence cyanelle RNase P, combining properties of bacterial and eukaryotic enzymes, occupies an intermediate position in RNA enzyme evolution. The first description of an RNA component in RNase P from a photosynthetic organelle might thus be an important step towards an understanding of plastid RNase P structure and function.
Base Sequence↗