Publications (10) View all
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Article: Diterpene cyclases and the nature of the isoprene fold.
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ABSTRACT: The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain approximately 20 alpha-helices and the same conserved "QW" and DxDD motifs as in triterpene cyclases, indicating the presence of a betagamma barrel structure. Plant diterpene cyclases have a similar catalytic motif and betagamma-domain structure together with a third, alpha-domain, forming an alphabetagamma structure, and in H(+)-initiated cyclases, there is an EDxxD-like Mg(2+)/diphosphate binding motif located in the gamma-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (betagamma) bacterial triterpene cyclases to (betagamma) bacterial and thence to (alphabetagamma) plant diterpene cyclases.Proteins Structure Function and Bioinformatics 08/2010; 78(11):2417-32. · 3.39 Impact Factor -
Article: A single residue switch for Mg(2+)-dependent inhibition characterizes plant class II diterpene cyclases from primary and secondary metabolism.
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ABSTRACT: Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg(2+) cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg(2+) binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated in some fashion. While further investigating class II diterpene cyclase catalysis, we discovered a conserved basic residue that seems to act as a counter ion to the DXDD motif, enhancing the ability of aspartic acid to carry out the requisite energetically difficult protonation of a carbon-carbon double bond and also affecting inhibitory Mg(2+) binding. Notably, this residue is conserved as a histidine in enzymes involved in gibberellin biosynthesis and as an arginine in those dedicated to secondary metabolism. Interchanging the identity of these residues is sufficient to switch the sensitivity of the parent enzyme to inhibition by Mg(2+). These striking findings indicate that this is a single residue switch for Mg(2+) inhibition, which not only supports the importance of this biochemical regulatory mechanism in limiting gibberellin biosynthesis, but the importance of its release, presumably to enable higher flux, into secondary metabolism.Journal of Biological Chemistry 07/2010; 285(27):20558-63. · 4.77 Impact Factor -
Article: Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases.
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ABSTRACT: The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.Proceedings of the National Academy of Sciences 04/2010; 107(16):7521-6. · 9.68 Impact Factor -
Article: Characterization and inhibition of a class II diterpene cyclase from Mycobacterium tuberculosis: implications for tuberculosis.
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ABSTRACT: Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.Journal of Biological Chemistry 08/2009; 284(35):23574-9. · 4.77 Impact Factor -
Article: The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase.
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ABSTRACT: Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis.Plant Molecular Biology 01/2006; 59(6):881-94. · 4.15 Impact Factor
