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1.  Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus 
Scientific Reports  2014;4:5469.
The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level.
doi:10.1038/srep05469
PMCID: PMC4073121  PMID: 24968921
2.  Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement 
Nature  2013;503(7477):10.1038/nature12643.
Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.
doi:10.1038/nature12643
PMCID: PMC3844076  PMID: 24162851
3.  Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1 
Nature chemical biology  2013;9(4):244-246.
We identify an Arabidopsis pyridoxal-phosphate (PLP)-dependent aminotransferase, VAS1, whose loss-of-function simultaneously increases levels of the phytohormone auxin and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). VAS1 uses the ethylene biosynthetic intermediate Met as an amino donor and the auxin biosynthetic intermediate, indole-3-pyruvic acid (3-IPA) as an amino acceptor to produce L-Trp and 2-oxo-4-methylthiobutyric acid (KMBA). Our data indicate that VAS1 serves key roles in coordinating the levels of these two vital hormones.
doi:10.1038/nchembio.1178
PMCID: PMC3948326  PMID: 23377040
4.  Evolution of the Chalcone Isomerase Fold from Fatty Acid-Binding to Stereospecific Enzyme 
Nature  2012;485(7399):10.1038/nature11009.
Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes1. However, the lineage of the enzyme chalcone isomerase (CHI) remained a quandary. In vascular plants, CHI-catalyzed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defense2, and development3. CHI operates near the diffusion limit with stereospecific control4,5. While associated primarily with plants, the CHI-fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties, and in vivo functional characterization of a non-catalytic CHI-fold family from plants. A. thaliana contains five actively transcribed CHI-fold genes, three of which additionally encode amino-terminal chloroplast-transit sequences (cTP). These three CHI-fold proteins localize to plastids, the site of de novo fatty acid (FA) biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core FA biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased FA storage in the developing embryo. In vitro, these proteins are Fatty Acid-binding Proteins (FAP). FAP knockout A. thaliana plants exhibit elevated alpha-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically ‘perfected’ enzyme6 from a non-enzymatic ancestor over a defined period of plant evolution.
doi:10.1038/nature11009
PMCID: PMC3880581  PMID: 22622584
5.  Chemodiversity in Selaginella: a reference system for parallel and convergent metabolic evolution in terrestrial plants 
Early plants began colonizing the terrestrial earth approximately 450 million years ago. Their success on land has been partially attributed to the evolution of specialized metabolic systems from core metabolic pathways, the former yielding structurally and functionally diverse chemicals to cope with a myriad of biotic and abiotic ecological pressures. Over the past two decades, functional genomics, primarily focused on flowering plants, has begun cataloging the biosynthetic players underpinning assorted classes of plant specialized metabolites. However, the molecular mechanisms enriching specialized metabolic pathways during land plant evolution remain largely unexplored. Selaginella is an extant lycopodiophyte genus representative of an ancient lineage of tracheophytes. Notably, the lycopodiophytes diverged from euphyllophytes over 400 million years ago. The recent completion of the whole-genome sequence of an extant lycopodiophyte, S. moellendorffii, provides new genomic and biochemical resources for studying metabolic evolution in vascular plants. 400 million years of independent evolution of lycopodiophytes and euphyllophytes resulted in numerous metabolic traits confined to each lineage. Surprisingly, a cadre of specialized metabolites, generally accepted to be restricted to seed plants, have been identified in Selaginella. Initial work suggested that Selaginella lacks obvious catalytic homologs known to be involved in the biosynthesis of well-studied specialized metabolites in seed plants. Therefore, these initial functional analyses suggest that the same chemical phenotypes arose independently more commonly than anticipated from our conventional understanding of the evolution of metabolism. Notably, the emergence of analogous and homologous catalytic machineries through convergent and parallel evolution, respectively, seems to have occurred repeatedly in different plant lineages.
doi:10.3389/fpls.2013.00119
PMCID: PMC3650682  PMID: 23717312
Selaginella; specialized metabolism; chemodiversity; parallel evolution; convergent evolution
6.  Discovery of a metabolic alternative to the classical mevalonate pathway 
eLife  2013;2:e00672.
Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature.
DOI: http://dx.doi.org/10.7554/eLife.00672.001
eLife digest
Living things make thousands of chemicals that are vital for life, and are also useful as medicines, perfumes, and food additives. The largest family of these natural chemicals is called the isoprenoids, and members of this family are found in all three domains of life: the eukaryotes (such as plants and animals), the Archaea (an ancient group of single-celled microbes), and bacteria.
The isoprenoids are made from a smaller building block called isopentenyl diphosphate, IPP for short, that contains five carbon atoms and two phosphate groups. IPP can be produced in two ways. The classical mevalonate pathway is found in most eukaryotes, including humans; statin drugs are used to inhibit this pathway to treat those with high cholesterol and reduce the risk of heart disease. The second pathway does not use the compound mevalonate and is found in many, but not all, bacteria as well as the chloroplasts of plants. Until recently, however, the enzymes needed for the last two steps of the classical mevalonate pathway appeared to be missing in the Archaea and in some bacteria.
Researchers subsequently discovered that an enzyme called isopentenyl phosphate kinase, shortened to IPK, was responsible for one of these two missing steps—the addition of IPP’s second phosphate group. The way this enzyme worked also suggested that there was an alternative mevalonate pathway in which the order of the last two steps was reversed. However, the identity of the enzyme responsible for the other step—the removal of a molecule of carbon dioxide to make the starting material needed by IPK—remained mysterious.
Now Dellas et al. have discovered the enzyme responsible for this missing step in Green non-sulphur bacteria, confirming the existence of the alternative mevalonate pathway for the first time. Previously it had been thought that this enzyme acted in the classical mevalonate pathway; but in fact this enzyme has evolved a new function and is not involved in the classical pathway at all. Moreover, Dellas et al. show that Green non-sulphur bacteria, and some eukaryotes, also have functional IPK enzymes. This means that IPK has now unexpectedly been observed in all three domains of life, and hints at another target to medically control mevalonate pathways. The discovery of the missing enzyme in the alternative pathway opens the door to the re-examination of many other living things, to find which have the new pathway and to work out why.
DOI: http://dx.doi.org/10.7554/eLife.00672.002
doi:10.7554/eLife.00672
PMCID: PMC3857490  PMID: 24327557
Mevalonate pathway; Isopentenyl diphosphate; Archaea; Mevalonate phosphate decarboxylase; Chloroflexi; Plants; Arabidopsis; Other
7.  Structural and kinetic analysis of prolyl-isomerization/phosphorylation cross-talk in the CTD code 
ACS chemical biology  2012;7(8):1462-1470.
The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. Ser2 and Ser5 are the major phosphorylation sites in vivo while Pro3 and Pro6 can adopt either cis or trans conformations. CTD regulation of transcription is mediated both by phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases.
Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but that have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.
doi:10.1021/cb3000887
PMCID: PMC3423551  PMID: 22670809
8.  Structure–function relationships in plant phenylpropanoid biosynthesis 
Current opinion in plant biology  2005;8(3):249-253.
Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. As an experimental tool set, structural biology provides a high-resolution means to uncover detailed information about the structure–function relationships of metabolic enzymes at the atomic level. Together with genomic and biochemical approaches and an appreciation of molecular evolution, structural enzymology holds great promise for addressing a number of questions relating to secondary or, more appropriately, specialized metabolism. Why is secondary metabolism so adaptable? How are reactivity, regio-chemistry and stereo-chemistry steered during the multi-step conversion of substrates into products? What are the vestigial structural and mechanistic traits that remain in biosynthetic enzymes during the diversification of substrate and product selectivity? What does the catalytic landscape look like as an enzyme family traverses all possible lineages en route to the acquisition of new substrate and/or product specificities? And how can one rationally engineer biosynthesis using the unique perspectives of evolution and structural biology to create novel chemicals for human use?
doi:10.1016/j.pbi.2005.03.013
PMCID: PMC2861907  PMID: 15860421
9.  Digging for answers, smelling a hint of success and tasting triumph 
Nature chemical biology  2007;3(11):690-691.
The C12 ‘earthy’ odorant geosmin is derived from the C15 metabolite farnesyl diphosphate. Metabolic transformation now seems to be catalyzed by a bifunctional protein having two operatively independent sesquiterpene synthase domains. The domains are catalytically linked through the passive diffusion of a C15 alcohol product of the N-terminal catalytic domain to the C-terminal catalytic domain for the final steps of geosmin formation.
doi:10.1038/nchembio1107-690
PMCID: PMC2859038  PMID: 17948014
10.  Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase 
Virology Journal  2012;9:305.
Background
Despite the effectiveness of highly active antiretroviral therapy (HAART), there remains an urgent need to develop new human immunodeficiency virus type 1 (HIV-1) inhibitors with better pharmacokinetic properties that are well tolerated, and that block common drug resistant virus strains.
Methods
Here we screened an in-house small molecule library for novel inhibitors of HIV-1 replication.
Results
An active compound containing a 3-aminoimidazo[1,2-a]pyridine scaffold was identified and quantitatively characterized as a non-nucleoside reverse transcriptase inhibitor (NNRTI).
Conclusions
The potency of this compound coupled with its inexpensive chemical synthesis and tractability for downstream SAR analysis make this inhibitor a suitable lead candidate for further development as an antiviral drug.
doi:10.1186/1743-422X-9-305
PMCID: PMC3560175  PMID: 23231773
HIV-1; NNRTI; Inhibitor
11.  Genetically Encoding Unnatural Amino Acids in Neural Stem Cells and Optically Reporting Voltage-sensitive Domain Changes in Differentiated Neurons 
Stem cells (Dayton, Ohio)  2011;29(8):1231-1240.
Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal and mammalian cells using orthogonal tRNA/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins.
doi:10.1002/stem.679
PMCID: PMC3209808  PMID: 21681861
Neural stem cells; unnatural amino acids; voltage sensing; fluorescence imaging
12.  Structural basis of steroid hormone perception by the receptor kinase BRI1 
Nature  2011;474(7352):467-471.
Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans intracellular receptors recognise these molecules. Plants however perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). The BRI1 ectodomain structure at 2.5 angstrom resolution reveals a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70 amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations closely map to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings have mechanistic implications for hormone, developmental and innate immunity signaling pathways in plants that use similar receptors.
doi:10.1038/nature10153
PMCID: PMC3280218  PMID: 21666665
13.  Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH4 hybrid cyclase 
Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography–mass spectrometry (GC–MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3 → CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25–4.64 for the CH3 → CH2 eliminations; (3) a significant intermolecular KIE (1.33 ± 0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1β position of epiaristolochene; (5) stereoselective eliminations of the 1β and 9β protons in formation of epiaristolochene and its Δ1(10) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1α proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.
doi:10.1016/j.abb.2005.09.002
PMCID: PMC2883252  PMID: 16309622
Sesquiterpenes; Eremophilanes; Spirovetivane; Germacrane; Enzyme mechanisms; Stereochemistry; Deuterium labeling; Isotope effects; Rearrangements; Cyclizations; Carbocations
14.  Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products 
Nature  2005;435(7044):983-987.
The anti-oxidant naphterpin is a natural product containing a polyketide-based aromatic core with an attached 10-carbon geranyl group derived from isoprenoid (terpene) metabolism1–3. Hybrid natural products such as naphterpin that contain 5-carbon (dimethylallyl), 10-carbon (geranyl) or 15-carbon (farnesyl) isoprenoid chains possess biological activities distinct from their non-prenylated aromatic precursors4. These hybrid natural products represent new anti-microbial, anti-oxidant, anti-inflammatory, anti-viral and anti-cancer compounds. A small number of aromatic prenyltransferases (PTases) responsible for prenyl group attachment have only recently been isolated and characterized5,6. Here we report the gene identification, biochemical characterization and high-resolution X-ray crystal structures of an architecturally novel aromatic PTase, Orf2 from Streptomyces sp. strain CL190, with substrates and substrate analogues bound. In vivo, Orf2 attaches a geranyl group to a 1,3,6,8-tetra-hydroxynaphthalene-derived polyketide during naphterpin biosynthesis. In vitro, Orf2 catalyses carbon–carbon-based and carbon–oxygen-based prenylation of a diverse collection of hydroxyl-containing aromatic acceptors of synthetic, microbial and plant origin. These crystal structures, coupled with in vitro assays, provide a basis for understanding and potentially manipulating the regio-specific prenylation of aromatic small molecules using this structurally unique family of aromatic PTases.
doi:10.1038/nature03668
PMCID: PMC2874460  PMID: 15959519
15.  Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase 
Nature chemical biology  2006;2(9):494-502.
Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two ~3,000-residue D. discoideum proteins, termed ‘steely’, as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.
doi:10.1038/nchembio811
PMCID: PMC2864586  PMID: 16906151
16.  Floral benzenoid carboxyl methyltransferases: From in vitro to in planta function 
Phytochemistry  2005;66(11):1211-1230.
Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT’s three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in planta depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses might represent the ancestor for the presently existing floral genes which during evolution gained different expression profiles and encoded enzymes with the ability to accept structurally similar substrates.
doi:10.1016/j.phytochem.2005.03.031
PMCID: PMC2864587  PMID: 15946712
17.  Structural Basis for the Modulation of CDK-Dependent/Independent Activity of Cyclin D1 
Cell cycle (Georgetown, Tex.)  2006;5(23):2760-2768.
D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target.
Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (1) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (2) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (3) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets.
Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle.
PMCID: PMC2864588  PMID: 17172845
cyclins; cyclin dependent kinase; in silico docking; CDK inhibitors; homology model
18.  Biosynthesis of Plant Volatiles: Nature’s Diversity and Ingenuity 
Science (New York, N.Y.)  2006;311(5762):808-811.
Plant volatiles (PVs) are lipophilic molecules with high vapor pressure that serve various ecological roles. The synthesis of PVs involves the removal of hydrophilic moieties and oxidation/hydroxylation, reduction, methylation, and acylation reactions. Some PV biosynthetic enzymes produce multiple products from a single substrate or act on multiple substrates. Genes for PV biosynthesis evolve by duplication of genes that direct other aspects of plant metabolism; these duplicated genes then diverge from each other over time. Changes in the preferred substrate or resultant product of PV enzymes may occur through minimal changes of critical residues. Convergent evolution is often responsible for the ability of distally related species to synthesize the same volatile.
doi:10.1126/science.1118510
PMCID: PMC2861909  PMID: 16469917
19.  A Soluble, magnesium-independent prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic substrates 
FEBS letters  2007;581(16):2889-2893.
Fnq26 from Streptomyces cinnamonensis DSM 1042 is a new member of the recently identified CloQ/Orf2 class of prenyltransferases. The enzyme was overexpressed in E. coli and purified to apparent homogeneity, resulting in a soluble, monomeric protein of 33.2 kDa. The catalytic activity of Fnq26 is independent of the presence of Mg2+ or other divalent metal ions. With flaviolin (2,5,7-trihydroxy-1,4-naphthoquinone) as substrate, Fnq26 catalyzes the formation of a carbon–carbon-bond between C-3 (rather than C-1) of geranyl diphosphate and C-3 of flaviolin, i.e. an unusual ‘‘reverse’’ prenylation. With 1,3-dihydroxynaphthalene and 4-hydroxybenzoate as substrates Fnq26 catalyzes O-prenylations.
doi:10.1016/j.febslet.2007.05.031
PMCID: PMC2860617  PMID: 17543953
Prenyltransferase; Reverse prenylation; Furanonaphthoquinone; Streptomyces
20.  Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis 
The Journal of biological chemistry  2005;280(34):30496-30503.
4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/ketoreductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase.
doi:10.1074/jbc.M502239200
PMCID: PMC2860619  PMID: 15970585
21.  Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases 
Summary
In plants, type I and II S-adenosyl-L-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56–58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 μM) and furfuryl thiol (Km 60 μM) were the best substrates (6–7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.
doi:10.1111/j.1365-313X.2006.02680.x
PMCID: PMC2860623  PMID: 16623883
Catharanthus roseus; S-methyltransferase; O-methyltransferase; evolution; protein modeling; homology-based cDNA cloning
22.  Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities 
Bioorganic & medicinal chemistry  2008;16(17):8117-8126.
NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon–carbon-based and carbon–oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.
doi:10.1016/j.bmc.2008.07.052
PMCID: PMC2860626  PMID: 18682327
Enzyme catalysis; Prenylation; Flavonoid; Polyketide; Kinetics
23.  The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene 
Summary
Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subspecies axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the man-made species P. hybrida, a hybrid of P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subspecies parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent-synthesizing and scent-emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide-like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame-shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by a yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNAi also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.
doi:10.1111/j.1365-313X.2009.03834.x
PMCID: PMC2860387  PMID: 19222805
pollination; plant volatile; scent; evolution; petunia
24.  Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes*S 
The Journal of biological chemistry  2004;280(5):3686-3696.
The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1β-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1β(OH)EA versus 3α(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1β(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1β(OH)EA, 2β(OH)EA, and 3β(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted ~1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1β(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1β(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1β(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.
doi:10.1074/jbc.M411870200
PMCID: PMC2859954  PMID: 15522862
25.  Structural Determinants and Modulation of Substrate Specificity in Phenylalanine-Tyrosine Ammonia-Lyases 
Chemistry & biology  2006;13(12):1327-1338.
Summary
Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an α,β-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for β-amino acid biosynthesis.
doi:10.1016/j.chembiol.2006.11.011
PMCID: PMC2859959  PMID: 17185228

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