Resume

• 2016

  Master of Business Administration - MBA

  Business Administration and Management

  General

  Purdue University

  Master of Business Administration (M.B.A.)

  Full-time MBA for STEM professionals (11 months)

  Finance

  General

  Corporate consultant

  Spring 2017 (Endocyte

  Inc).\nCertificate in Manufacturing & Technology Management

  Global Supply Chain Management Initiative.\nBeta Gamma Sigma business honorary society.

  Purdue University - Krannert School of Management

  RapiData: A Practical Course in Macromolecular X-ray Diffraction Measurement at Brookhaven National Laboratory

  Infrared

  Near Infrared and Raman Spectroscopy at Bruker

• 2008

  Purdue University

  Honolulu

  HI

  Research in carotenoid uptake by cultured cells with Dr Robert Cooney.

  Researcher

  University of Hawaii Cancer Center

  West Lafayette

  Indiana

  Assistant Professor

  Department of Biochemistry

  Principal Investigator

  Purdue University

  Lafayette

  Indiana Area

  Scientific

  financial

  and competitive assessment of emerging opportunities in molecular diagnostics and allied areas. Activities: grant proposals (PI and contributor)

  recruitment

  operations

  analysis

  intelligence gathering

  external collaboration.

  Director Of Technology

  Amplified Sciences

  LLC

  West Lafayette

  IN

  Visiting Scholar

  Purdue University

• 2000

  Washington University in St. Louis

  Saint Louis

  MO

  Assistant Professor

  Department of Chemistry

  Principal Investigator

  Washington University in St. Louis

  member

  Protein Society

  American Society for Microbiology

  A 501(c)(3) charity that supports the annual Midwest Enzyme Chemistry Conference (MECC).

  Treasurer

  Friends of the Midwest Enzyme Chemistry Conference

  English

  Richard C. Wolfgang Prize for Outstanding Academic Work in Chemistry

  Yale University

  American Institute of Chemists Award

  American Institute of Chemists

  National Cancer Institute (NCI) Cancer Research Training Postdoctoral Fellowship

  National Cancer Institute/National institutes of Health (NCI/NIH)

  CAREER award

  The Faculty Early Career Development (CAREER) Program is a [National Science] Foundation-wide activity that offers the National Science Foundation's most prestigious awards in support of early-career faculty who have the potential to serve as academic role models in research and education and to lead advances in the mission of their department or organization.

  National Science Foundation

  Herbert C. Krannert Fellowship

  Krannert School of Management

  Purdue University

  Howard Hughes Medical Institute Predoctoral Fellowship

  Howard Hughes Medical Institute (HHMI)

  Beta Gamma Sigma Honorary

  Business honorary

  awarded for graduating In the top 5% or so.

• 1996

  Massachusetts Institute of Technology (MIT)

  University of Hawaii Cancer Center

  Cambridge

  MA

  NCI Fellow performing research on de novo purine biosynthesis in the group of Prof JoAnne Stubbe

  Postdoctoral Researcher

  Massachusetts Institute of Technology (MIT)

  West Lafayette

  iN

  Consultant

  Amplified Sciences

  LLC

• 1989

  Doctor of Philosophy (Ph.D.)

  Howard Hughes Medical Institute Predoctoral Fellow

  Richard Wolfgang Prize for best doctoral thesis in chemistry

  Dissertation title: \"Recombinant phenylalanine hydroxylase overexpression and kinetic characterization. Structural

  mechanistic

  and spectroscopic studies of its allosteric activation and active site iron reduction processes.\"

  Chemistry

  Yale University

• 1988

  member

  American Chemical Society

  International business honor society

  member

  Beta Gamma Sigma

• 1985

  Bachelor’s Degree

  Chemistry

  Research with Prof J. T. Hupp (Chemistry) on mixed-valence bimetallic complexes; summer support from NSF-REU fellowship. \nRubber Teeth magazine.

  Northwestern University

• 1976

  High School

  Ka Punahou newspaper editor-in-chief

  Ka Punahou

  Punahou School

• Organized and led the 35th Annual Midwest Enzyme Chemistry Conference (MECC) held 12 Sep 2015 at the Illinois Institute of Technology (IIT).

  Midwest Enzyme Chemistry Conference

  Treasurer

  Manager for 501(c)(3) nonprofit devoted to sustaining the Midwest Enzyme Chemistry Conference

  held annually in Chicago.

  Friends of the Midwest Enzyme Chemistry Conference

  Food Pantry volunteer

  St. John's/Lafayette Urban Ministry food programs provide year-round emergency food assistance to the greater Lafayette community.

  St John's Episcopal Church

  Science

  Life Sciences

  Biochemistry

  Enzymes

  Chemistry

  Scientific Leadership

  Research

  Protein Purification

  Data Analysis

  Protein Expression

  Microbiology

  Protein Chemistry

  You are lost without a map: navigating the sea of protein structures

  Nicholas R. Silvaggi

  Highlights\n• Protein crystal structures are not photographs

  they are models based on data.\n• All models require subjective interpretation of electron density maps.\n• Even excellent structures have ambiguous regions; often the most interesting parts. Prudent structure users always look at the electron density.\n\nAbstract\n\nX-ray crystal structures propel biochemistry research like no other experimental method

  since they answer many questions directly and inspire new hypotheses. Unfortunately

  many users of crystallographic models mistake them for actual experimental data. Crystallographic models are interpretations

  several steps removed from the experimental measurements

  making it difficult for nonspecialists to assess the quality of the underlying data. Crystallographers mainly rely on “global” measures of data and model quality to build models. Robust validation procedures based on global measures now largely ensure that structures in the Protein Data Bank (PDB) are largely correct. However

  global measures do not allow users of crystallographic models to judge the reliability of “local” features in a region of interest. Refinement of a model to fit into an electron density map requires interpretation of the data to produce a single “best” overall model. This process requires inclusion of most probable conformations in areas of poor density. Users who misunderstand this can be misled

  especially in regions of the structure that are mobile

  including active sites

  surface residues

  and especially ligands. This article aims to equip users of macromolecular models with tools to critically assess local model quality. Structure users should always check the agreement of the electron density map and the derived model in all areas of interest

  even if the global statistics are good. We provide illustrated examples of interpreted electron density as a guide for those unaccustomed to viewing electron density.

  You are lost without a map: navigating the sea of protein structures

  Elwood A. Mullins

  Journal of Bacteriology

  Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations

  among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase

  which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA

  reliance upon nucleotide pools

  and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA

  a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation

  which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti

  a CO2 loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes

  therefore

  support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.

  A specialized citric acid cycle requiring succinyl-coenzyme A (CoA): acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti

  Linda C. Kurz

  Biochemistry

  Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent

  slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA

  the initial product. Using stopped-flow and steady-state fluorescence methods

  kinetic substrate isotope effects

  and mutagenesis of active site residues

  we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate

  acetyl-CoA

  also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the nonhydrolyzable product of the condensation reaction

  dethiacitryl-CoA

  forms a particularly stable complex with TpCS but not pig heart CS.

  The partial substrate dethiaacetyl-coenzyme A mimics all critical carbon acid reactions in the condensation half-reaction catalyzed by Thermoplasma acidophilum citrate synthase

  Stefano Donini

  Acta Cryst. (2015). F71

  1292-1299\ndoi:10.1107/S2053230X15015939\n\nCitrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA)

  and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that

  when compared with several liganded TpCS structures

  helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag

  the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand

  the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.\nKeywords: thermophile; euryarchaeon; conformation change; carbon-carbon bond formation.

  An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

  Jose M Sanchez-Ruiz

  Arne Holmgren

  Masaru Tanokura

  Sergi Garcia-Manyes

  Pallav Kosuri

  Jorge Alegre-Cebollada

  Inmaculada Sanchez-Romero

  Zi-Ming Zhao

  Alvaro Inglés-Prieto

  Raul Perez-Jimenez

  ABSTRACT: It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction

  based on sequence predicted by phylogenetic analysis

  of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes

  and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate

  we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged

  they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.

  Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

  Michael E. Johnson

  Loredana C. Huma

  Elwood A. Mullins

  Kelly L. Sullivan

  Analytical Biochemistry

  The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however

  no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h

  as assessed by the Bratton–Marshall assay (BMA) for diazotizable amines. The ZnSO4 stopping reagent is proposed to chelate CAIR

  enabling delayed analysis of this acid-labile product by BMA or other HTS methods.\nKeywords: Purine biosynthesis; Aminoimidazole; Substrate depletion; Chelation

  Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

  A new synthetic method allows incorporation of 13C or 15N into selected positions within purine nucleotide bases

  starting from simple labeled precursors. The procedure harnesses diverse enzymes to support biosynthesis by the pentose phosphate and de novo purine pathways. Selective isotope incorporation should expand the range of RNAs that are amenable to NMR analysis.\n\nPoint of view on: Schultheisz

  H. L.

  Szymczyna

  B. R.

  Scott

  L. G.

  and Williamson

  J. R. 2008 Pathway engineered enzymatic de novo purine nucleotide synthesis ACS Chem. Biol. 3 499 511

  The purine machine scores a base hit

  Jeong-Won Nam

  Aaron E. Ransome

  Sasitorn Sivanuntakorn

  ABSTRACT: Acetobacter aceti converts ethanol to acetic acid

  and strains highly resistant to both are used to make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification

  which implies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase (AaCS)

  a hexameric type II citrate synthase

  is required for acetic acid resistance and

  therefore

  would be expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequence of strong selective pressure to function at low pH

  and unexpectedly high thermal stability for a protein that has evolved to function at ∼30 °C. The crystal structure of AaCS

  complexed with oxaloacetate (OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX)

  was determined to 1.85 Å resolution using protein purified by a tandem affinity purification procedure. This is the first crystal structure of a “closed” type II CS

  and its active site residues interact with OAA and CMX in the same manner observed in the corresponding type I chicken CS·OAA·CMX complex. While AaCS is not regulated by NADH

  it retains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface of AaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic charges than EcCS; this constellation of charged residues is stable in varied pH environments and may be advantageous in the A. aceti cytoplasm.\n

  Structure of an NADH-insensitive hexameric citrate synthase that resists acid inactivation

  Elwood A. Mullins

  Acetic Acid Bacteria

  Vinegar production requires acetic acid bacteria that produce

  tolerate

  and conserve high levels of acetic acid. When ethanol is depleted

  aerobic acetate overoxidation to carbon dioxide ensues. The resulting diauxic growth pattern has two logarithmic growth phases

  the first associated with ethanol oxidation and the second associated with acetate overoxidation. The vinegar factory isolate Acetobacter aceti strain 1023 has a long intermediate stationary phase that persists at elevated acetic acid levels. Strain 1023 conserves acetic acid despite possessing a complete set of citric acid cycle (CAC) enzymes

  including succinyl-CoA:acetate CoA-transferase (SCACT)

  the product of the acetic acid resistance (aar) gene aarC. In this study

  cell growth and acid production were correlated with the functional expression of aargenes using reverse transcription-polymerase chain reaction

  Western blotting

  and enzyme activity assays. Citrate synthase (AarA) and SCACT (AarC) were abundant in A. aceti strain 1023 during both log phases

  suggesting the transition to acetate overoxidation was not a simple consequence of CAC enzyme induction. A mutagenized derivative of strain 1023 lacking functional AarC readily oxidized ethanol but was unable to overoxidize acetate

  indicating that the CAC is required for acetate overoxidation but not ethanol oxidation. The primary role of the aar genes in the metabolically streamlined industrial strain A. aceti 1023 appears to be to harvest energy via acetate overoxidation in otherwise depleted medium.\n\nComments: This article was published in Acetic Acid Bacteria (2013)

  2(1). doi: http://dx.doi.org/10.4081/aab.2013.s1.e3.\n\nThis publication also references the following datasets:\nDOI: 10.4231/D31834278\nDOI: 10.4231/D3WH2DF2W\n\nKeywords: AarA

  SixA

  AarC

  citric acid cycle

  acetate overoxidation\n

  Functional analysis of the acetic acid resistance (aar) gene cluster in Acetobacter aceti strain 1023

  Aaron E. Ransome

  Jacob Schaefer

  ABSTRACT: Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid

  but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes

  most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex

  the citrate central carboxylate C6 13C peak moves upfield

  indicating diminution of negative charge

  and broadens

  indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Nδ-unprotonated (pyridine-like) form and an Nδ-protonated (pyrrole-like) form

  each of which is ∼4 Å from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Nδ that are invoked in the class I PurE mechanism.\n

  Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase (PurE) detected by REDOR NMR

  Brittney Z. Heard

  Kelly M. MacArthur

  Abstract: The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 Å resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange

  a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH

  neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters

  a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm

  and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function

  protein structure and dynamics

  and side-chain ionization.

  Atomic -resolution crystal structure of Acetobacter aceti thioredoxin

  a thiol-disulfide exchange catalyst that functions at low pH

  Lee Sael

  Courtney M. Starks

  Elwood A. Mullins

  Protein Science

  Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor

  UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties

  other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a “speckled” charge decoration

  in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern

  which is a distinctive feature of A. aceti proteins

  creates a stabilizing electrostatic network without stiffening the protein or compromising protein–solvent interactions.

  Formyl‐coenzyme A (CoA): oxalate CoA‐transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability

  ABSTRACT: Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms

  forming distinct tetrahedral intermediates <3 Å apart. In this study

  crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294

  indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a

  an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate

  in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride

  even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  Functional dissection of the bipartite active site of CoA-transferase acetate:succinyl-CoA CoA-transferase

  Elwood A. Mullins

  Biochemistry

  Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates

  using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts. Mechanistic studies of class I CoA-transferases suggested that acyl-CoA binding energy is used to accelerate rate-limiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate [White

  H.

  and Jencks

  W. P. (1976) J. Biol. Chem. 251

  1688–1699]. The class I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cycle in Acetobacter aceti. In an effort to identify residues involved in substrate recognition

  X-ray crystal structures of a C-terminally His6-tagged form (AarCH6) were determined for several wild-type and mutant complexes

  including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a closed complex containing dethiaacetyl-CoA

  which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases.

  Crystal structures of Acetobacter aceti succinyl-coenzyme A (CoA):acetate CoA-transferase reveal specificity determinants and illustrate the mechanism used by class I CoA-transferases

  Kelly L. Sullivan

  Elwood A. Mullins

  PloS ONE

  Many food plants accumulate oxalate

  which humans absorb but do not metabolize

  leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA

  which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria

  including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT)

  YfdU (OXC)

  and YfdE

  a class III CoA-transferase that is 30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification

  in vitro characterization

  2.1-Å crystal structure

  and functional assignment of YfdE. YfdE and UctC

  an orthologue from the obligate aerobe Acetobacter aceti

  perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive

  conserved active site loops (the glycine-rich loop and the GNxH loop

  respectively) that appear to encode substrate specificity.

  Function and X-ray crystal structure of Escherichia coli YfdE

  Richard K. Wilson

  Jyothi Thimmapuramd

  Lucinda Fulton

  Aaron E. Ransome

  Ketaki Bhide

  Vincent Magrini

  Sandra W. Clifton

  The genome sequence of Acetobacter aceti 1023

  an acetic acid bacterium\nadapted to traditional vinegar fermentation

  comprises 3.0 Mb (chromosome plus plasmids).\nA. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but\npossesses many additional insertion sequence elements.

  Draft genome sequence of Acetobacter aceti strain 1023

  a vinegar factory isolate

  Susan C. Hockings

  Courtney M. Starks

  Sylvain Tranchimand

  Biochemistry

  De novo purine biosynthesis proceeds by two divergent paths. In bacteria

  yeasts

  and plants

  5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N5-carboxy-AIR (N5-CAIR) synthetase (PurK) and N5-CAIR mutase (class I PurE). In animals

  the conversion of AIR to CAIR requires a single enzyme

  AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO2

  respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene

  the hallmarks of CO2-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase

  the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO2 but not N5-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence

  the TdPurE·AIR complex contains a portal that appears to allow the CO2 substrate to enter the active site. In the human pathogen T. denticola

  purine biosynthesis should depend on available CO2 levels. Because spirochetes lack carbonic anhydrase

  the corresponding reduction in bicarbonate demand may confer a selective advantage.

  Treponema denticola PurE is a bacterial AIR carboxylase

  JoAnne Stubbe

  Steven E. Ealick

  Akimitsu Okamoto

  Paul Peng

  Timothy E. Barder

  Judith B. Zaugg

  Mariya Morar

  Aaron A. Hoskins

  ABSTRACT: N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis

  a pH−rate profile

  DFT calculations

  and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved

  protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5‘-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart

  implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR

  a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common

  buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.

  N5-CAIR mutase: Role of a CO2 binding site and substrate movement in catalysis

  Jeong-Won Nam

  ABSTRACT: Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Δ12 position of linoleic acid

  forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR)

  acetylenase transcripts were identified in non-seed C. alpina tissues

  which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds

  genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5)

  or that compete for substrate (FAD3)

  were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels

  and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 104-fold higher acetylenase expression in developing seeds than in flower heads. Collectively

  the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Δ12 acetylenase in a polyacetylene biosynthetic pathway

  so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product

  crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes.

  Cloning and transcriptional analysis of Crepis alpina fatty acid desaturases affecting the biosynthesis of crepenynic acid

  SUMMARY: Like many promising drug targets

  phosphoethanolamine methyltransferase is part of a pathway that is present in a pathogen but not in mammalian hosts. In this issue of Structure

  Lee and Jez describe a structure of this phospholipid biosynthesis enzyme from a parasitic nematode and reveal a reconfigured active site.

  A biosynthetic enzyme worms its way out of a conserved mechanism

  Enzymes are amazing chemical catalysts. Working alongside great students

  my research on enzymes expanded the toolkit for biotechnology

  synthetic biology

  and bioprocess/agricultural chemistry. Today my goal is to make new enzyme-based products.

  T. J. (Joe)

  Kappock

  Amplified Sciences

  LLC