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PubMed2004

Mimicking enzyme evolution by generating new (betaalpha)8-barrels from (betaalpha)4-half-barrels.

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (betaalpha)8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (betaalpha)4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (betaalpha)8-barrels from (betaalpha)4-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N'[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (betaalpha)4-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the beta-barrel of HisF-CC, the monomeric and compact (betaalpha)8-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (betaalpha)4-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (betaalpha)8-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (betaalpha)8-barrels with novel functions might have evolved by the exchange of (betaalpha)4-half-barrel domains with distinct functional properties.

Aldose-Ketose Isomerases↗
PubMed2001

Directed enzyme evolution.

Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.

Bacterial Proteins↗
PubMed1998

Analogous enzymes: independent inventions in enzyme evolution.

It is known that the same reaction may be catalyzed by structurally unrelated enzymes. We performed a systematic search for such analogous (as opposed to homologous) enzymes by evaluating sequence conservation among enzymes with the same enzyme classification (EC) number using sensitive, iterative sequence database search methods. Enzymes without detectable sequence similarity to each other were found for 105 EC numbers (a total of 243 distinct proteins). In 34 cases, independent evolutionary origin of the suspected analogous enzymes was corroborated by showing that they possess different structural folds. Analogous enzymes were found in each class of enzymes, but their overall distribution on the map of biochemical pathways is patchy, suggesting multiple events of gene transfer and selective loss in evolution, rather than acquisition of entire pathways catalyzed by a set of unrelated enzymes. Recruitment of enzymes that catalyze a similar but distinct reaction seems to be a major scenario for the evolution of analogous enzymes, which should be taken into account for functional annotation of genomes. For many analogous enzymes, the bacterial form of the enzyme is different from the eukaryotic one; such enzymes may be promising targets for the development of new antibacterial drugs.

Amino Acid Sequence↗
PubMed2005

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.

Bacterial Proteins↗
PubMed2002

Glutamate decarboxylase: computer studies of enzyme evolution.

The homology of subunit primary sequence of 40 glutamate decarboxylases (GAD) of different origin was analyzed by multiple alignment. A phylogenetic tree was designed on the basis of the resulting data. The following groups are distinguished in the consensus tree: archeans, bacteria, plant eukaryotes, and animal eukaryotes. The latter are clearly divided into two branches according to two enzyme isoforms. Borders of PLP domains in each enzyme were detected. The consensus phylogenetic tree for PLP domains is structurally rather similar to that obtained for subunits. Twenty homologous motifs of from 15 to 87 amino acid residues were revealed in all GAD studied. The results revealed the division of all of the enzymes into groups with characteristic sets of motifs in each and a fixed order of their arrangement along the sequence. Thus, we can show the divergent evolution of the enzyme. The results of multiple alignments during structural analysis of the 40 GAD confirmed and extended our previous data on conserved residues that arrange the position of the coenzyme (PLP) in the enzyme active center. The following residues should be noted: lysine forming a Schiff base with the PLP aldehyde group, an adjacent histidine, and aspartic acid that establishes a link with nitrogen of the PLP pyridine ring. The homology of the primary sequence fragments was also found in the residues in contact with the PLP phosphate group. Comparison of the GAD amino acid sequence with that of another PLP enzyme, aspartate aminotransferase, revealed a binding site for carboxylic group of the substrate--glutamic acid. The structures carrying out a particular catalytic function of all GAD studied were detected, i.e., convergent evolution of the enzyme was revealed.

Amino Acid Sequence↗
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↗
PubMed1995

Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme.

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0.11 h-1 (td = 6.3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M(r)) of the native GDH was 110,000. SDS-PAGE resulted in one single band that represented a polypeptide with a M(r) of 28,000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4.2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗
PubMed1984

Construction of hybrid genes of 6-aminohexanoic acid-oligomer hydrolase and its analogous enzyme. Estimation of the intramolecular regions important for the enzyme evolution.

Hybrids were constructed of the genes for two homologous enzymes, 6-aminohexanoic acid-oligomer hydrolase (EII, one of the nylon oligomer degradation enzymes), and its probable evolutionary antecedent (EII'). The structural genes of EII (nylB) and EII' (nylB') have 88% similarity in their nucleotide sequences, an open frame encoding a peptide of 392 amino acids, conserved restriction sites, and in vitro recombination between these genes at the corresponding restriction sites generated genes directing various hybrid enzymes. In a comparison of the EII, EII', and the hybrid enzymes, we concluded that one or more of the four amino acid alterations that occurred in the intramolecular region (between amino acids 162-257) of EII' is essential to the adaptation of the enzyme to nylon oligomer degradation, and that its effect is enhanced 20-fold by one or more further alterations in the 258-380 region. Our results also suggest that this technique is useful for improving enzyme characteristics.

Amidohydrolases↗
PubMed2000

Enzyme evolution explained (sort of).

Sites in proteins evolve at markedly different rates; some are highly conserved, others change rapidly. We have developed a maximum likelihood method to identify regions of a protein that evolve rapidly or slowly relative to the remaining structure. We also show that solvent accessibility and distance from the catalytic site are major determinants of evolutionary rate in eubacterial isocitrate dehydrogenases. These two variables account for most of the rate heterogeneity not ascribable to stochastic effects.

Catalytic Domain↗
PubMed2006

Influence of metabolic network structure and function on enzyme evolution.

BACKGROUND: Most studies of molecular evolution are focused on individual genes and proteins. However, understanding the design principles and evolutionary properties of molecular networks requires a system-wide perspective. In the present work we connect molecular evolution on the gene level with system properties of a cellular metabolic network. In contrast to protein interaction networks, where several previous studies investigated the molecular evolution of proteins, metabolic networks have a relatively well-defined global function. The ability to consider fluxes in a metabolic network allows us to relate the functional role of each enzyme in a network to its rate of evolution. RESULTS: Our results, based on the yeast metabolic network, demonstrate that important evolutionary processes, such as the fixation of single nucleotide mutations, gene duplications, and gene deletions, are influenced by the structure and function of the network. Specifically, central and highly connected enzymes evolve more slowly than less connected enzymes. Also, enzymes carrying high metabolic fluxes under natural biological conditions experience higher evolutionary constraints. Genes encoding enzymes with high connectivity and high metabolic flux have higher chances to retain duplicates in evolution. In contrast to protein interaction networks, highly connected enzymes are no more likely to be essential compared to less connected enzymes. CONCLUSION: The presented analysis of evolutionary constraints, gene duplication, and essentiality demonstrates that the structure and function of a metabolic network shapes the evolution of its enzymes. Our results underscore the need for systems-based approaches in studies of molecular evolution.

Amino Acid Substitution↗
PubMed2004

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology.

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA3, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA14, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.

Biotechnology↗
PubMed2004

Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways.

3-Carboxy-cis,cis-muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the beta-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-muconate to muconolactones. We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution. PpCMLE is a homotetramer and belongs to the fumarase class II superfamily. The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers. It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members. However, this charge relay pair is broken in PpCMLE. The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE. The structures of prokaryotic MLEs and that of CMLE from the eukaryotic Neurospora crassa are completely different from that of PpCMLE, indicating MLEs and CMLEs, as well as the prokaryotic and eukaryotic CMLEs, evolved from distinct ancestors, although they catalyze similar reactions. The structural differences may be related to recognition by substrates and to differences in the mechanistic pathways by which these enzymes catalyze their respective reactions.

Amino Acid Sequence↗
PubMed1996

Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway.

A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.

Acetylation↗
PubMed2026

An activity-resistance trade-off constrains enzyme evolution.

The presence of self-resistance genes in antibiotic-producing organisms poses a paradox: How can resistance evolve before the antibiotic exists, and how can an antibiotic producer arise without first evolving resistance? Here, we examine the evolutionary origins of self-resistance to mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). The MPA biosynthetic gene cluster (BGC) includes a resistant IMPDH-B. Homologs of IMPDH-B occur not only in MPA producers but also in many nonproducing fungi, where remnants of the MPA BGC remain detectable. The phylogeny of IMPDH-B is incongruent with the fungal species tree, consistent with multiple horizontal gene transfer events between Aspergillus and Sordariomycetes. We characterized eleven extant IMPDH-Bs, five from MPA producers and six from nonproducers, along with seven resurrected ancestral enzymes (Anc1-Anc7). MPA resistance appeared between Anc2 and Anc3 and coincided with a loss of catalytic efficiency. Across both ancestral and extant enzymes, MPA resistance correlated strongly with reduced activity, revealing a robust activity-resistance trade-off that has persisted for millions of years. Unexpectedly, both the IMPDH-Bs and ancestral enzymes Anc3-Anc7 were also resistant to ribavirin-5'-monophosphate (RVP), an IMP-competitive inhibitor. Because MPA and RVP bind to similar enzyme conformations, the activity-resistance trade-off may reflect a design constraint imposed by the need to maintain resistance to multiple inhibitors. Intriguingly, although Anc1 and Anc2 are equally sensitive to MPA, Anc2 shows reduced susceptibility to RVP. This pattern suggests that preexisting resistance to another IMPDH inhibitor may have created a permissive background for the later evolution of MPA biosynthesis.

IMP Dehydrogenase↗
PubMed1997

Directed evolution of enzyme catalysts.

Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution in vitro includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.

Biotechnology↗