An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Aindrila Mukhopadhyay,1,6* Edward Baidoo,1,6 Kelly Bender5,6 (bender@micro.siu.edu), Peter Benke,1,6 Swapnil Chhabra,1,6 Elliot Drury,3,6 Masood Hadi,2,6 Zhili He,4,6 Jay Keasling1,6 (keasling@.berkeley.edu), Kimberly Keller,3,6 Eric Luning,1,6 Francesco Pingitore,1,6 Alyssa Redding,1,6 Jarrod Robertson,3,6 Rajat Sapra,2,6 Anup Singh2,6 (aksingh@sandia.gov), Judy Wall3,6 (wallj@ missouri.edu), Grant Zane,3,6 Aifen Zhou,4,6 and Jizhong Zhou4,6 (jzhou@rccc.ou.edu)

Measurements of low molecular weight metabolites have been increasingly incorporated in the characterization of cellular physiology, qualitative studies in functional genomics, and stress response determination. The application of cutting edge analytical technologies to the measurement of metabolites and the changes in metabolite concentrations under defined conditions have helped illuminate the effects of perturbations in pathways of interest, such as the tricarboxylic acid cycle, as well as unbiased characterizations of microbial stress responses as a whole. Owing to the complexity of microbial metabolite extracts and the large number of metabolites therein, advanced and high-throughput separation techniques in gas chromatography, liquid chromatography, and capillary electrophoresis have been coupled to mass spectrometry usually high-resolution mass spectrometry, but not exclusively - to make these measurements.

DOE oversees 350 cleanup projects involving soil contaminated with metals/radionuclides. The life-cycle cost of these projects is at least $220 billion over 70 years, without breakthroughs. A thorough understanding of the biogeochemistry, especially stress responses in metal/radionuclide bacteria, enables prediction of natural attenuation and new strategies for remediation saving DOE billions in cleanup, risk assessment, and environmental stewardship. This application is representative of an array of environmental, ecological, and bioenergy stewardship challenges that rest on developing a detailed understanding of environmental microbial physiology, community interactions, population genetics and functions and ultimately evolution. The diversity of knowledge/technology necessary to accomplish these goals necessitates a team science approach, building a sophisticated experimental and computational infrastructure.

Aindrila Mukhopadhyay,1,6* Edward Baidoo,1,6 Kelly Bender5,6 (bender@micro.siu.edu), Peter Benke,1,6 Swapnil Chhabra,1,6 Elliot Drury,3,6 Masood Hadi,2,6 Zhili He,4,6 Jay Keasling1,6 (keasling@.berkeley.edu), Kimberly Keller,3,6 Eric Luning,1,6 Francesco Pingitore,1,6 Alyssa Redding,1,6 Jarrod Robertson,3,6 Rajat Sapra,2,6 Anup Singh2,6 (aksingh@sandia.gov), Judy Wall3,6 (wallj@ missouri.edu), Grant Zane,3,6 Aifen Zhou,4,6 and Jizhong Zhou4,6 (jzhou@rccc.ou.edu)

The fragmentation characteristics of protonated alanylglycylglycine, [AGG + H](+), were investigated by tandem mass spectrometry in MALDI-TOF/TOF, ion trap, and hybrid sector instruments. b(2) is the most abundant fragment ion in MALDI-TOF/TOF, ion trap, and hybrid sector metastable ion (MI) experiments, while y(2) is slightly more abundant than b(2) in collision activated dissociation (CAD) performed in the sector instrument. The A-G amide bond is cleaved on the a(1)-y(2) pathway resulting in a proton-bound dimer of GG and MeCH=NH. Depending on the fragmentation conditions employed, this dimer can then (1) be detected as [AGG + H - CO](+), (2) dissociate to produce y(2) ions, [GG + H](+), (3) dissociate to produce a(1) ions, [MeCH=NH + H](+), or (4) rearrange to expel NH(3) forming a [AGG + H - CO - NH(3)](+) ion. The activation method and the experimental timescale employed largely dictate which of, and to what extent, these processes occur. These effects are qualitatively rationalized with the help of quantum chemical and RRKM calculations. Two mechanisms for formation of the [AGG + H - CO - NH(3)](+) ion were evaluated through nitrogen-15 labeling experiments and quantum chemical calculations. A mechanism involving intermolecular nucleophilic attack and association of the GG and imine fragments followed by ammonia loss was found to be more energetically favorable than expulsion of ammonia in an S(N)2-type reaction.

ABSTRACT Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13 C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the ( S )-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.

Carl Abulencia, Eric J. Alm, Gary Anderson, Edward Baidoo, Peter Benke, Sharon Borglin, Eoin L. Brodie, Romy Chakraborty, Swapnil Chhabra, Gabriela Chirica, Dylan Chivian, Michael J. Cipriano, M.E. Clark, Paramvir S. Dehal, Elliot C. Drury, Inna Dubchak, Dwayne A. Elias, Matthew W. Fields, J. Gabster, Sara P. Gaucher, Jil Geller, B. Giles, Masood Hadi, Terry C. Hazen, Qiang He, Zhili He, Christopher L. Hemme, E. Hendrickson, Kristina L. Hillesland, Hoi-Ying Holman, Katherine H. Huang, Y. Wayne Huang, C. Hwang, Janet Jacobsen, Marcin P. Joachimiak, Dominique C. Joyner, Jay D. Keasling, Keith Keller, Martin Keller, J. Leigh, T. Lie, Aindrila Mukhopadhyay, Richard Phan, Francesco Pingitore, Morgan Price, Alyssa M. Redding, Joseph A. Ringbauer Jr., Rajat Sapra, Christopher W. Schadt, Amy Shutkin, Anup K. Singh, David A. Stahl, Sergey M. Stolyar, Yinjie Tang, Joy D. Van Nostrand, Chris B. Walker, Judy D. Wall, Eleanor Wozei, Zamin K. Yang, Huei-Che Yen, Grant Zane, Aifen Zhou, Jizhong Zhou, and Adam P. Arkin* (aparkin@lbl.gov)

Fluxes through known metabolic pathways and the presence of novel metabolic reactions are often determined by feeding isotopically labeled substrate to an organism and then determining the isotopomer distribution in amino acids in proteins. However, commonly used techniques to measure the isotopomer distributions require derivatization prior to analysis (gas chromatography/mass spectrometry (GC/MS)) or large sample sizes (nuclear magnetic resonance (NMR) spectroscopy). Here, we demonstrate the use of Fourier transform-ion cyclotron resonance mass spectrometry with direct infusion via electrospray ionization to rapidly measure the amino acid isotopomer distribution in a biomass hydrolysate of the soil bacterium Desulfovibrio vulgaris Hildenborough. By applying high front-end resolution for the precursor ion selection followed by sustained off-resonance irradiation collision-induced dissociation, it was possible to determine exactly and unambiguously the specific locations of the labeled atoms in the amino acids, which usually requires a combination of 2-D 13C NMR spectroscopy and GC/MS. This method should be generally applicable to all biomass samples and will allow more accurate determination of metabolic fluxes with less work and less sample.

The Li+ complexes of the isomeric α-dipeptide radicals H2NCHC(O)NHCH2COOH (GlyGly) and H2NCH2C(O)NHCHCOOH (GlyGly) are formed in the gas phase from the isomeric complexes [PheGly+Li]+ and [GlyPhe+Li]+, respectively, via homolytic cleavage of the corresponding benzyl side chains. The isomers undergo distinctively different reactions upon collisionally activated dissociation (CAD) and, hence, represent unique, non-interconverting species. The investigation of deuterated isotopomers and of dipeptide radicals with Ala residues permits complete elucidation of the dissociation pathways of the radical complexes. The majority of reactions observed are promoted by the radical site, with the location of the unpaired electron playing an important role in the types of reactions taking place. Analogous differences are found for dilithiated complexes of GlyGly and GlyGly, in which the COOH termini are derivatized to COO−Li+ salt bridges. Density functional theory calculations confirm that the lithiated and dilithiated α-dipeptide radicals have distonic character; the radical is largely localized on the N- or C-terminal α-C atom and the charge is largely localized on the metal ions. In the most stable conformers, the Li+ ion(s) are bound between the amide carbonyl and C-terminal carbonyl (or carboxylate) groups. Theory predicts a higher thermodynamic stability for the complexes of the N-terminal radical GlyGly, as reflected by the significantly higher yield, with which these complexes are formed (from their PheGly precursors), compared to the GlyGly complexes.

An alpha, omega-dihydroxypoly(fluorooxetane) was prepared from a fluorinated oxetane monomer and characterized by matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) and gel permeation chromatographic (GPC) methods. Estimated molecular weights were compared to those derived from NMR spectroscopic end-group analysis. The results of 2-D NMR spectroscopic analysis agreed well with 1-D NMR end-group analytical results and MALDI MS experiments. Because of the small molecular weight of the product, the results from the mass spectroscopic and NMR methods compared favorably. The overestimated molecular weights obtained from GPC are explained by aggregation of the amphiphilic poly(fluorooxetane) in a relatively poor solvent. The robustness of the methods was verified by fractionation of the polymer and subsequent analysis of the fractions by MALDI MS. In addition, information regarding product architecture and copolymerization with THF, which was used to complex the BF3 catalyst, was obtained using tandem mass spectrometry (MS/MS) methods.

Published Ahead of Print 17 November 2006. 2007, 189(3):940. DOI: 10.1128/JB.00948-06. J. Bacteriol. Richard Phan, Terry C. Hazen and Jay D. Keasling Yinjie Tang, Francesco Pingitore, Aindrila Mukhopadhyay, Resonance Mass Spectrometry and Fourier Transform-Ion Cyclotron Gas Chromatography-Mass Spectrometry Hildenborough using Desulfovibrio vulgaris Central Metabolic Pathways in Pathway Confirmation and Flux Analysis of

The Li+ complexes of the isomeric dipeptide pairs PheGly/GlyPhe, PheAla/AlaPhe, and TrpAla/AlaTrp, namely, [Pep + Li]+, and of the corresponding lithium carboxylates, namely, [Pep - H + 2Li]+, are produced in the gas phase by desorption ionization, and their unimolecular chemistry is probed by tandem mass spectrometry experiments at various activation conditions. At low internal energies, monolithiated isomers dissociate to the same products, formed through a mixed anhydride intermediate in which the sequence information is lost. Isomerization to the mixed anhydride is less competitive at higher internal energies, which start promoting sequence-specific fragmentations. On the other hand, dilithiated isomers (they contain a permanent COO-Li+ salt bridge) do not rearrange to an anhydride and give rise to substantially different fragmentation patterns; structurally diagnostic c1- and y1-type fragments are observed at all internal energies, allowing for unequivocal sequence assignment. The mono- and dilithiated peptides undergo loss of their aromatic side chain to form distonic radical ions carrying Li+ charge(s) and one unpaired electron at an alpha-C atom of the peptide backbone. The yield of such metal-bound peptide radicals is particularly high from the dilithiated complexes, [Pep - H + 2Li]+. Upon activation, the Li+ ions become mobile and can be shuttled to the various basic sites of the dipeptides, where they may initiate backbone fragmentation or the elimination of small neutral molecules.

The elimination of carbon monoxide and water from a series of protonated dipeptides, [XxxYyy + H] + , is investigated by tandem mass spectrometry experiments and density functional theory. The combined results show that CO loss occurs on the a 1 -y 1 pathway, which begins by rearrangement of the added proton to the amide N-atom and creates the proton-bound dimer of an amino acid (Yyy) and an imine (that from Xxx residue). The loss of H 2 O is initiated from a tautomer in which the added proton has migrated to the hydroxyl group of the C-terminus, thereby promoting the formation of an ion with protonated oxazolone structure (a nominal b 2 ion). The highest yields of [XxxYyy+H−CO] + and [XxxYyy+H−H 2 O] + are observed at threshold energies. As the internal energy of the protonated dipeptides increases, these primary products are depleted by consecutive dissociations yielding mostly backbone fragments. Specifically, [XxxYyy+H−CO] + decomposes to y 1 (protonated Yyy) and a 1 (immonium ion of Xxx residue), while [XxxYyy+H−H 2 O] + produces a 2 and the immonium ions of residues Xxx (a 1 ) and Yyy (“internal” immonium ion). Water loss takes place more efficiently when the more basic residue is at the C-terminal position. Increasing the basicity of the N-terminal residue enhances the extent of CO versus H 2 O loss and introduces the competitive elimination of NH 3 . The dissociations leading to eliminations of small neutrals (CO, H 2 O, etc.) generally proceed over transition states that lie higher in energy than the corresponding dissociation products. The excess energy is disposed of either in translational or rovibrational modes of the products, depending on the stability of the incipient noncovalent assemblies emerging during the cleavage of the small neutrals.

Silver(I)-2,6-bis(ethanolimidazolemethyl)pyridine hydroxide (4a) and silver(I)-2,6-bis(propanolimidazolemethyl)pyridine hydroxide (4b) are water-soluble silver(I)-carbene complexes that were synthesized in high yield by reacting silver(I) oxide with N-substituted pincer ligands 3 (a = 2,6-bis(ethanolimidazoliummethyl)pyridine diiodide, b = 2,6-bis(propanolimidazoliummethylpyridine)pyridine dibromide). The X-ray crystal structure of 4a is a one-dimensional linear polymer, whereas the mass spectroscopy confirms a monomer in the gas phase. A change in the anion of 4a from a hydroxide to a hexafluorophosphate formed a silver(I)-carbene complex 4c that is dimeric in structure and insoluble in water. The bactericidal activities of the water-soluble silver(I)-carbene complexes were found to be improved over that of silver nitrate.

Author(s): Keasling, Jay D. | Abstract: The microorganism Desulfovibrio vulgaris Hildenborough , because of its metabolic versatility, its ability to remediate metals and radionuclides, and the ease with which it can be maintained in culture is of particular interest to the DOE. However, the effective implementation of remediation strategies and the use of natural attenuation for the cleanup of DOE sites is dependant upon understanding critical chemical, physical, and biological processes. Thus, an understanding of regulatory mechanisms and cellular responses to different environmental factors affecting the metal remediation activity in situ is of great importance. However, understanding the genome, transcriptome, and proteome may not be enough to fully characterize the pathways involved in bioremediation. For example, the proteome cannot be completely predicted from the transcriptome because of post-translational modifications, some of which may occur slowly, while others occur rapidly irrespective of the rate of protein synthesis. Furthermore, transcriptome changes can be significantly slower than changes to the proteome. An approach based solely on transcriptomics may also be inadequate, since there are many genes that are not under transcriptional control. Whereas the metabolome is further down the line from gene function and so reflects more closely the activities of a cell at the functional level. Capillary electrophoresis and time of flight mass spectrometry (CE-TOFMS) is generally considered a promising technique for metabolome analysis due to its high separation efficiency and accurate mass determination with low sample volume requirements. We have utilized novel CE-TOFMS methods for the detection and identification of anionic and cationic metabolites within D. vulgaris at mid-log phase. This approach has proved successful in determining most types of compound, including amino acids, carbohydrate derivatives, coenzyme As, nucleosides, nucleotides and organic acids.