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1.  Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates 
PLoS ONE  2013;8(8):e72372.
Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using 31P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.
doi:10.1371/journal.pone.0072372
PMCID: PMC3756997  PMID: 24009677
2.  Pyruvate is the source of the two carbons that are required for formation of the imidazoline ring of 4-demethylwyosine 
Biochemistry  2011;50(49):10573-10575.
TYW1 catalyzes the condensation of N-methylguanosine with two carbon atoms from an unknown second substrate to form 4-demethylwyosine, which is a common intermediate in the biosynthesis of all of the hypermodified RNA bases related to wybutosine found in eukaryal and archaeal tRNAPhe. Of potential substrates examined, only incubation with pyruvate resulted in formation of 4-demethylwyosine. Moreover, incubation with C1, C2, C3, or C1,2,3-13C-labeled pyruvate showed that C2 and C3 are incorporated while C1 is not. The mechanistic implications of these results are discussed in the context of the structure of TYW1.
doi:10.1021/bi2015053
PMCID: PMC3232322  PMID: 22026549
3.  Surface-Induced Dissociation Reveals the Quaternary Substructure of a Heterogeneous Non-Covalent Protein Complex 
Analytical Chemistry  2011;83(8):2862-2865.
As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within non-covalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of non-covalent complexes, few developments have been made towards the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here we illustrate the activation and dissociation by surface-induced dissociation of a heterohexamer, toyocamycin nitrile hydratase, directly into its constituent trimers. We propose that the single-step nature of this activation in combination with high energy deposition allows for dissociation prior to significant unfolding or other large-scale rearrangement. This method can potentially allow for dissociation of a protein complex into subcomplexes facilitating the mapping of subunit contacts and thus determination of quaternary structure of protein complexes.
doi:10.1021/ac200452b
PMCID: PMC3343771  PMID: 21417466
Mass spectrometry; non-covalent protein complexes; surface-induced dissociation; quaternary structure
4.  Delivery of tailor-made cobalamin to methylmalonyl-CoA mutase 
Nature chemical biology  2008;4(3):158-159.
Methylmalonyl coenzyme A mutase (MCM) catalyzes the adenosylcobalamin-dependent isomerization of methylmalonyl-CoA to succinyl-CoA. Adenosyltransferase, an enzyme that carries out the final step in biosynthesis of adenosylcobalamin, is shown to be involved in delivery of the cofactor to MCM.
doi:10.1038/nchembio0308-158
PMCID: PMC3227859  PMID: 18277972
5.  E. coli QueD is a 6-carboxy-5,6,7,8-tetrahydropterin synthase† 
Biochemistry  2009;48(11):2301-2303.
To elucidate the early steps required during biosynthesis of a broad class of 7-deazapurine containing natural products, we have studied the reaction catalyzed by Escherichia coli QueD, a 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase (PTPS) homolog possibly involved in queuosine biosynthesis. While mammalian PTPS homologs convert 7,8-dihydroneopterin triphosphate (H2NTP) to 6-pyruvoyltetrahydropterin (PPH4) in biopterin biosynthesis, E. coli QueD catalyzes the conversion of H2NTP to 6-carboxy-5,6,7,8-tetrahydropterin (CPH4). E. coli QueD can also convert PPH4 and sepiapterin to CPH4, allowing a mechanism to be proposed.
doi:10.1021/bi9001437
PMCID: PMC3227869  PMID: 19231875
6.  Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor† 
Biochemistry  2006;45(39):12144-12155.
The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/ mol of protein and utilizes guanosine 5′-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074–15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912–36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.
doi:10.1021/bi061005x
PMCID: PMC3227873  PMID: 17002314
7.  The deazapurine biosynthetic pathway revealed: In vitro enzymatic synthesis of preQ0 from guanosine-5′-triphosphate in four steps† 
Biochemistry  2009;48(18):3847-3852.
Deazapurine-containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogs found throughout biology including various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as antibiotic, antiviral, and antineoplastic agents, the biosynthetic route toward deazapurine production has remained largely elusive for more than 40 years. Here we present the first in vitro preparation of the deazapurine nucleoside, preQ0, by the successive action of four enzymes. The pathway includes the conversion of the recently identified biosynthetic intermediate, 6-carboxy-5,6,7,8-tetrahydropterin, to a novel intermediate, 7-carboxy-7-deazaguanine (CDG), by an unusual transformation catalyzed by B. subtilis QueE, a member of the radical SAM enzyme superfamily. The carboxylate moiety on CDG is converted subsequently to a nitrile to yield preQ0 by either B. subtilis QueC or S. rimosus ToyM in an ATP-dependent reaction, in which ammonia serves as the nitrogen source. The results presented here are consistent with early radiotracer studies on deazapurine biosynthesis and provide a unified pathway for the production of deazapurines in nature.
doi:10.1021/bi900400e
PMCID: PMC2693876  PMID: 19354300
8.  Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor  
The X-ray crystal structure of a 6-pyruvoyltetrahydropterin synthase homolog of unknown function has been determined at 1.5 Å resolution. The protein retains residues required for pterin binding, but nearly all catalytic residues are missing.
The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.
doi:10.1107/S1744309108027048
PMCID: PMC2564891  PMID: 18931427
SCO 6650; Streptomyces coelicolor; 6-pyruvoyltetrahydropterin synthase homolog
9.  Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor 
The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.
doi:10.1107/S1744309108027048
PMCID: PMC2564891  PMID: 18931427
10.  ROSETTA STONE FOR DECIPHERING DEAZAPURINE BIOSYNTHESIS: PATHWAY FOR PYRROLOPYRIMIDINE NUCLEOSIDES TOYOCAMYCIN AND SANGIVAMYCIN 
Chemistry & biology  2008;15(8):790-798.
Summary
Pyrrolopyrimidine nucleosides analogs, collectively referred to as deazapurines, are an important class of structurally diverse compounds found in a wide variety of biological niches. In this report, a cluster of genes from Streptomyces rimosus involved in production of the deazapurine antibiotics sangivamycin and toyocamycin was identified using forward genetics methods. The cluster includes toyocamycin nitrile hydratase, an enzyme that catalyzes the conversion of toyocamycin to sangivamycin. In addition to this rare nitrile hydratase, the cluster encodes a GTP cyclohydrolase I, linking the biosynthesis of deazapurines to folate biosynthesis, and a set of purine salvage genes, which presumably convert the guanine moiety from GTP to the adenine-like deazapurine base found in toyocamycin and sangivamycin. The gene cluster presented here could potentially serve as a “Rosetta stone” to inform on deazapurine biosynthesis in other bacterial species.
doi:10.1016/j.chembiol.2008.07.012
PMCID: PMC2603307  PMID: 18721750
11.  Probing Nitrogen Sensitive Steps in the Free Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase 
The contribution of C-N bond-breaking/making steps to the rate of the free-radical-mediated deamination of vicinal amino alcohols by adenosylcobalamin-dependent ethanolamine ammonia-lyase has been investigated by 15N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15N IE's were determined for three substrates, ethanolamine, (R)-2-aminopropanol, and (S)-2-aminopropanol using isotope ratio mass spectrometry analysis of the product ammonia. Measurements with all three substrates gave measurable, normal 15N IE's; however, the IE of (S)-2-aminopropanol was ∼ 5-fold greater than the other two. Reaction mixtures frozen during the steady-state show that the 2-aminopropanols give EPR spectra characteristic of the initial substrate radical whereas ethanolamine gives spectra consistent with a product-related radical [Warncke, K.; Schmidt, J. C.; Kee, S.-C., J. Am. Chem. Soc. 1999, 121, 10522-10528]. The steady-state concentration of the radical with (R)-2-aminopropanol is ∼ half that observed with the S isomer, and with (R)-2-aminopropanol the steady-state level of radical is further reduced upon deuteration at C1. The results show that relative heights of kinetic barriers differ among the three substrates such that levels or identities of steady-state intermediates differ. 15N-Sensitive steps are significant contributors to V/K with (S)-2-aminopropanol.
doi:10.1021/ja060710q
PMCID: PMC2505056  PMID: 16734439
12.  The Copper-Inducible cin Operon Encodes an Unusual Methionine-Rich Azurin-Like Protein and a Pre-Q0 Reductase in Pseudomonas putida KT2440▿  
Journal of Bacteriology  2007;189(14):5361-5371.
The genome sequences of several pseudomonads have revealed a gene cluster containing genes for a two-component heavy metal histidine sensor kinase and response regulator upstream of cinA and cinQ, which we show herein to encode a copper-containing azurin-like protein and a pre-Q0 reductase, respectively. In the presence of copper, Pseudomonas putida KT2440 produces the CinA and CinQ proteins from a bicistronic mRNA. UV-visible spectra of CinA show features at 439, 581, and 719 nm, which is typical of the plastocyanin family of proteins. The redox potential of the protein was shown to be 456 ± 4 mV by voltametric titrations. Surprisingly, CinQ is a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the conversion of pre-Q0 to pre-Q1 in the nucleoside queuosine biosynthetic pathway. Gene disruptions of cinA and cinQ did not lead to a significant increase in the copper sensitivity of P. putida KT2440 under the conditions tested. Possible roles of CinA and CinQ to help pseudomonads adapt and survive under prolonged copper stress are discussed.
doi:10.1128/JB.00377-07
PMCID: PMC1951875  PMID: 17483220

Results 1-12 (12)