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1.  The Alpha Subunit of Nitrile Hydratase Is Sufficient for Catalytic Activity and Post-Translational Modification 
Biochemistry  2014;53(24):3990-3994.
Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV–visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.
PMCID: PMC4075990  PMID: 24914472
2.  Radical mediated ring formation in the biosynthesis of the hypermodified tRNA base wybutosine 
Wyosine and its derivatives are highly modified, acid labile tricyclic bases found at position 37 of tRNAPhe in archaea and eukarya. The formation of the common 4-demethylwyosine structural feature entails condensation of pyruvate and N-methylguanosine catalyzed by TYW1. This review will focus on the mechanism of this complex radical mediated transformation.
PMCID: PMC4320972  PMID: 23856057
3.  Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism 
Nature chemical biology  2013;10(2):106-112.
7-Carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg2+-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the first crystal structure of a QueE, along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg2+, which directly coordinates to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified (β6/α3) protein core and an expanded cluster-binding motif CX14CX2C.
PMCID: PMC3939041  PMID: 24362703
Radical enzymes; X-ray crystallography; adenosylmethionine; tetrahydropterin; adenosylcobalamin; deazapurine
4.  Spectroscopic, steady-state kinetic, and mechanistic characterization of the radical SAM enzyme QueE, which catalyzes a complex cyclization reaction in the biosynthesis of 7-deazapurines 
Biochemistry  2012;52(1):188-198.
7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from Bacillus subtilis to delineate the mechanism of conversion of CPH4 to CDG. QueE is a member of the radical S-adenosyl-L-methionine (SAM) superfamily, all of which use a bound [4Fe-4S]+ cluster to catalyze the reductive cleavage of SAM cofactor to generate methionine and a 5′-deoxyadenosyl radical (5′-dAdo•), which initiates enzymatic transformations requiring H-atom abstraction. The UV-visible, EPR, and Mössbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a Km of 20 ± 7 μM for CPH4 and kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp for the divalent metal ion of 0.21 ± 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH4 isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5′-dAdo• as initiating the ring contraction reaction catalyzed by QueE by abstraction of the H-atom from C-6 of CPH4.
PMCID: PMC4022186  PMID: 23194065
5.  Biosynthesis of pyrrolopyrimidines 
Bioorganic chemistry  2012;43:15-25.
Pyrrolopyrimidine containing compounds, also known as 7-deazapurines, are a collection of purine-based metabolites that have been isolated from a variety of biological sources and have diverse functions which range from secondary metabolism to RNA modification. To date, nearly 35 compounds with the common 7-deazapurine core structure have been described. This article will illustrate the structural diversity of these compounds and review the current state of knowledge on the biosynthetic pathways that give rise to them.
PMCID: PMC4022189  PMID: 22382038
Biosynthesis of 7-Deazapurines; 7-deazapurines in tRNA; 7-deazapurine in secondary metabolism
6.  Radical SAM enzymes involved in the biosynthesis of purine-based natural products 
Biochimica et biophysica acta  2012;1824(11):1245-1253.
The radical S-adenosyl-L-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5’-deoxyadenosyl radical, which is produced by reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F420.
PMCID: PMC4022190  PMID: 22902275
7.  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.
PMCID: PMC3756997  PMID: 24009677
8.  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.
PMCID: PMC3232322  PMID: 22026549
9.  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.
PMCID: PMC3343771  PMID: 21417466
Mass spectrometry; non-covalent protein complexes; surface-induced dissociation; quaternary structure
10.  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.
PMCID: PMC3227859  PMID: 18277972
11.  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.
PMCID: PMC3227869  PMID: 19231875
12.  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.
PMCID: PMC3227873  PMID: 17002314
13.  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.
PMCID: PMC2693876  PMID: 19354300
14.  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.
PMCID: PMC2564891  PMID: 18931427
SCO 6650; Streptomyces coelicolor; 6-pyruvoyltetrahydropterin synthase homolog
15.  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.
PMCID: PMC2564891  PMID: 18931427
Chemistry & biology  2008;15(8):790-798.
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.
PMCID: PMC2603307  PMID: 18721750
17.  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.
PMCID: PMC2505056  PMID: 16734439
18.  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.
PMCID: PMC1951875  PMID: 17483220

Results 1-18 (18)