Search our Database of Scientific Publications and Authors

I’m looking for a

    Details and Download Full Text PDF:
    Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

    BMC Evol Biol 2015 Jun 10;15:107. Epub 2015 Jun 10.
    Evolution of Metabolic Diversity, Unidad de Genómica Avanzada (Langebio), Cinvestav-IPN, Km 9.6 Libramiento Norte, Carretera Irapuato - León, CP 36821, Irapuato, México.
    Background: Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families.

    Results: We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2) predicted to adopt a (βα)6-fold, and thus entirely lacking a C-terminus phosphate-binding site, was identified and shown to have HisA activity.

    Conclusion: As expected, reconstruction of the evolution of PriA from HisA with HMM profiles suggest that functional shifts involve mutations in evolutionarily intermediate enzymes of otherwise functionally essential residues or motifs. These results are in agreement with a link between promiscuous enzymes and intragenic epistasis. HMM provides a convenient approach for gaining insights into these evolutionary processes.
    PDF Download - Full Text Link
    ( Please be advised that this article is hosted on an external website not affiliated with
    Source Status ListingPossible

    Similar Publications

    Co-occurrence of analogous enzymes determines evolution of a novel (βα)8-isomerase sub-family after non-conserved mutations in flexible loop.
    Biochem J 2016 05 29;473(9):1141-52. Epub 2016 Feb 29.
    Evolution of Metabolic Diversity Laboratory, Unidad de Genómica Avanzada (Langebio), Cinvestav-IPN, Irapuato, CP36821, México
    We investigate the evolution of co-occurring analogous enzymes involved in L-tryptophan and L-histidine biosynthesis in Actinobacteria Phylogenetic analysis of trpF homologues, a missing gene in certain clades of this lineage whose absence is complemented by a dual-substrate HisA homologue, termed PriA, found that they fall into three categories: (i) trpF-1, an L-tryptophan biosynthetic gene horizontally acquired by certain Corynebacterium species; (ii) trpF-2, a paralogue known to be involved in synthesizing a pyrrolopyrrole moiety and (iii) trpF-3, a variable non-conserved orthologue of trpF-1 We previously investigated the effect of trpF-1 upon the evolution of PriA substrate specificity, but nothing is known about the relationship between trpF-3 and priA After in vitro steady-state enzyme kinetics we found that trpF-3 encodes a phosphoribosyl anthranilate isomerase. However, mutation of this gene in Streptomyces sviceus did not lead to auxothrophy, as expected from the biosynthetic role of trpF-1 Biochemical characterization of a dozen co-occurring TrpF-2 or TrpF-3, with PriA homologues, explained the prototrophic phenotype, and unveiled an enzyme activity trade-off between TrpF and PriA. X-ray structural analysis suggests that the function of these PriA homologues is mediated by non-conserved mutations in the flexible L5 loop, which may be responsible for different substrate affinities. Read More
    Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation.
    PLoS Genet 2016 Jan 29;12(1):e1005836. Epub 2016 Jan 29.
    Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany.
    Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. Read More
    Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis.
    Proc Natl Acad Sci U S A 2011 Mar 14;108(9):3554-9. Epub 2011 Feb 14.
    European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse 85, D-22603 Hamburg, Germany.
    In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific single-substrate enzymes (HisA and TrpF), sharing a similar (β/α)(8)-barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Read More
    Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.
    Mol Biol Evol 2013 Sep 25;30(9):2024-34. Epub 2013 Jun 25.
    Evolution of Metabolic Diversity Laboratory, Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Cinvestav-IPN, Irapuato, México.
    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. Read More