PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-19 (19)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
more »
1.  Structures of Fluoro, Amino and Thiol Inhibitors Bound to the [Fe4S4] Protein IspH** 
doi:10.1002/anie.201208469
PMCID: PMC3734547  PMID: 23307751
Bioinorganic chemistry; Metalloenzymes; Terpenoides; IspH; LytB
2.  Discovery of acetylene hydratase activity of the iron–sulphur protein IspH 
Nature communications  2012;3:1042.
The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by the iron–sulphur enzyme IspH, producing the universal precursors of terpenes: isopentenyl diphosphate and dimethylallyl diphosphate. Here we report an unforeseen reaction discovered during the investigation of the interaction of IspH with acetylene inhibitors by X-ray crystallography, Mößbauer, and nuclear magnetic resonance spectroscopy. In addition to its role as a 2H+/2e− reductase, IspH can hydrate acetylenes to aldehydes and ketones via anti-Markovnikov/Markovnikov addition. The reactions only occur with the oxidised protein and proceed via η1-O-enolate intermediates. One of these is characterized crystallographically and contains a C4 ligand oxygen bound to the unique, fourth iron in the 4Fe-4S cluster: this intermediate subsequently hydrolyzes to produce an aldehyde product. This unexpected side to IspH reactivity is of interest in the context of the mechanism of action of other acetylene hydratases, as well as in the design of antiinfectives targeting IspH.
doi:10.1038/ncomms2052
PMCID: PMC3745992  PMID: 22948824
3.  O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase 
The Journal of organic chemistry  2012;77(14):6239-6261.
Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction.
doi:10.1021/jo3010364
PMCID: PMC3444172  PMID: 22780198
4.  Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation 
Journal of the American Chemical Society  2012;134(27):11225-11234.
The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and 2H, 17O, and 57Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin: thioredoxin reductase, in that one electron is transferred to the [4Fe-4S]2+ cluster, which then performs a formally two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate’s 3-CH2OH group has rotated away from the reduced iron-sulfur cluster, and the next, η3-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogs (4 and 5) follow the same reaction mechanism.
doi:10.1021/ja303445z
PMCID: PMC3394908  PMID: 22687151
5.  Virtual Screening, Selection and Development of a Benzindolone Structural Scaffold for Inhibition of Lumazine Synthase 
Bioorganic & medicinal chemistry  2010;18(10):3518-3534.
Virtual screening of a library of commercially available compounds vs. the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a Ki of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π–π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.
doi:10.1016/j.bmc.2010.03.072
PMCID: PMC2868945  PMID: 20430628
Mycobacterium tuberculosis; lumazine synthase; inhibitor; virtual screening
6.  Mechanistic insights on riboflavin synthase inspired by selective binding of the 6,7-dimethyl-8-ribityllumazine exomethylene anion 
Riboflavin synthase catalyzes the transfer of a 4-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of riboflavin synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4+2 cycloaddition affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
doi:10.1021/ja908395r
PMCID: PMC2832097  PMID: 20143812
Riboflavin synthase; biosynthesis of riboflavin; reaction mechanism; hydride transfer; cycloaddition
7.  Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis  
Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding.
The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R cryst = 23.7% (R free = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.
doi:10.1107/S0907444910029690
PMCID: PMC2935281  PMID: 20823551
Bacillus anthracis; riboflavin biosynthesis; lumazine synthase; anthrax; inhibition; drug design
8.  Discovery and Development of a Small Molecule Library with Lumazine Synthase Inhibitory Activity 
The Journal of organic chemistry  2009;74(15):5123-5134.
(E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2,4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100,000 compound library. The Ki of 9 vs. Mycobacterium tuberculosis lumazine synthase was 95 μM. Compound 9 is a structural analog of the lumazine synthase substrate, 5-amino-6-(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a Ki of 3.7 μM vs. M. tuberculosis lumazine synthase.
doi:10.1021/jo900238q
PMCID: PMC2760403  PMID: 19552377
9.  Improvement of the quality of lumazine synthase crystals by protein engineering 
Site-directed mutagenesis has been applied to improve the overexpression and purification of the icosahedral enzyme lumazine synthase from B. subtilis as well as to produce a new crystal form. The mutant protein crystallizes in space group R3 and diffracts X-rays to 1.6 Å resolution.
Icosahedral macromolecules have a wide spectrum of potential nanotechno­logical applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 Å icosahedral capsid consisting of 60 subunits and crystallizes in space group P6322 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 Å resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 Å, α = β = 90.0, γ = 120°, and diffracts to 1.6 Å resolution.
doi:10.1107/S1744309108015728
PMCID: PMC2443968  PMID: 18607092
lumazine synthase; icosahedral capsid; site-directed mutagenesis; crystal quality
10.  15N{31P} REDOR NMR Studies of the Binding of Phosphonate Reaction Intermediate Analogues to Saccharomyces cerevisiae Lumazine Synthase 
Biochemistry  2008;47(52):13942-13951.
Lumazine synthase catalyzes the reaction of 5-amino-6-D-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with (S)-3,4-dihydroxybutanone 4-phosphate (2) to afford 6,7-dimethyl-8-D-ribityllumazine (3), the immediate biosynthetic precursor of riboflavin. The overall reaction implies a series of intermediates that are incompletely understood. The 15N{31P} REDOR NMR spectra of three metabolically stable phosphonate reaction intermediate analogues complexed to Saccharomyces cerevisiae lumazine synthase have been obtained at 7 and 12 T. Distances from the phosphorus atoms of the ligands to the side chain nitrogens of Lys92, His97, Arg136, and His148 have been determined. These distances were used in combination with the X-ray crystal coordinates of one of the intermediate analogues complexed with the enzyme in a series of distance-restrained molecular dynamics simulations. The resulting models indicate mobility of the Lys92 side chain, which could facilitate the exchange of inorganic phosphate eliminated from the substrate in one reaction, with the organic phosphate-containing substrate necessary for the next reaction.
PMCID: PMC2630582  PMID: 19117095
11.  Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase – ligand recognition in a template for antimicrobial drug discovery 
The Febs Journal  2008;275(11):2779-2794.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α-phosphate not the binding site for the methyl-d-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.
doi:10.1111/j.1742-4658.2008.06418.x
PMCID: PMC2655357  PMID: 18422643
enzyme–ligand complex; GHMP kinase; isoprenoid biosynthesis; molecular recognition; non-mevalonate pathway
12.  Design, Synthesis, and Biochemical Evaluation of 1,5,6,7-Tetrahydro-6,7-dioxo-9-D-Ribitylaminolumazines Bearing Alkyl Phosphate Substituents as Inhibitors of Lumazine Synthase and Riboflavin Synthase 
The Journal of organic chemistry  2005;70(20):8162-8170.
The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. In order to obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analog inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with Ki values in the low nanomolar range. The additional carbonyl in the dioxolumazine system vs. the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase.
doi:10.1021/jo051332v
PMCID: PMC2548293  PMID: 16277343
13.  A Novel Lumazine Synthase Inhibitor Derived from Oxidation of 1,3,6,8-Tetrahydroxy-2,7-naphthyridine to a Tetraazaperylenehexaone Derivative 
The Journal of organic chemistry  2007;72(8):2769-2776.
Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-Ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66 ± 13 μM in Tris buffer and 22 ± 4 μM in phosphate buffer. This is significantly more potent than the reactant (Ki 350 ± 76 μM, competitive inhibition), which had previously been identified as a lumazine synthase inhibitor by high-throughput screening. Ab initio calculations indicate that the meso form is slightly less stable than the enantiomeric form, and that the two forms interconvert rapidly at room temperature.
doi:10.1021/jo062246d
PMCID: PMC2526313  PMID: 17348709
14.  Evolution of Vitamin B2 Biosynthesis: 6,7-Dimethyl-8-Ribityllumazine Synthases of Brucella 
Journal of Bacteriology  2006;188(17):6135-6142.
The penultimate step in the biosynthesis of riboflavin (vitamin B2) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high Km for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg−1 min−1. Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.
doi:10.1128/JB.00207-06
PMCID: PMC1595393  PMID: 16923880
15.  Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers 
BMC Biochemistry  2005;6:24.
Background
Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15N/13C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-13C6]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15N-phenylalanine.
Results
Quantitative NMR analysis showed incorporation of the proffered [U-13C6]glucose into the ribose moiety of ribonucleosides (40 – 45%) and into the amino acids, alanine (41%), glutamic acid/glutamine (C-4 and C-5, 30%) and aspartate/asparagine (15%). Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%). Prior to the incorporation into protein the proffered 15N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent.
Conclusion
Growth of S. frugiperda cells in the presence of [U-13C6]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15N-labelled amino acids may be hampered by loss of the 15N-label by transamination.
doi:10.1186/1471-2091-6-24
PMCID: PMC1310531  PMID: 16285881
16.  Characterization of the Saccharomyces cerevisiae Fol1 Protein: Starvation for C1 Carrier Induces Pseudohyphal Growth 
Molecular Biology of the Cell  2004;15(8):3811-3828.
Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Δ deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5′-formyltetrahydrofolate). Growth of the fol1Δ deletion strain on folinic acid–supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.
doi:10.1091/mbc.E03-09-0680
PMCID: PMC491839  PMID: 15169867
17.  Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments 
BMC Biochemistry  2003;4:18.
Background
Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.
Results
A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.
Conclusion
Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.
doi:10.1186/1471-2091-4-18
PMCID: PMC337094  PMID: 14690539
18.  Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle 
Journal of Bacteriology  2001;183(14):4305-4316.
In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO2 molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-13C2]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C1 units derived therefrom and not into other cell compounds.
doi:10.1128/JB.183.14.4305-4316.2001
PMCID: PMC95321  PMID: 11418572
19.  Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii 
Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling.
doi:10.1021/jz4023738
PMCID: PMC3977573  PMID: 24723998

Results 1-19 (19)