Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety.

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non-heme diiron enzyme VlmB in a reaction in which the N-N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting µ-oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB-catalyzed reaction represents the first example of enzyme-catalyzed azoxy formation and is expected to proceed by an atypical mechanism.

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N-Nitroglycine by Variovorax sp. Strain JS1663.

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (nnlA) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed ß-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives.IMPORTANCE The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N-Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the isolation of a Gram-negative soil bacterium, Variovorax sp. strain JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a ß-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help identify other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives.

Naturally-occurring nitro compounds.

Naturally-occurring nitro compounds display great structural diversity, and a wide range of biological activities. This review summarizes current information on the structures of naturally-occurring nitro compounds and on the biosynthesis of the nitro group.

Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA.

The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.

Streptomyces scabies 87-22 contains a coronafacic acid-like biosynthetic cluster that contributes to plant-microbe interactions.

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway.

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N-methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.

Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP).

Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the -35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA-vlmH and vlmI-vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.

Identification, characterization, and bioconversion of a new intermediate in valanimycin biosynthesis.

The antibiotic valanimycin is a naturally occurring azoxy compound isolated from Streptomyces viridifaciens. Detailed investigations have shown that valanimycin is derived from L-valine and L-serine via the intermediacy of O-(L-seryl)isobutylhydroxylamine. Sequence analysis of the valanimycin biosynthetic genes provides relatively few clues concerning the nature of the later stages of the pathway. Two exceptions are provided by the vlmJ and vlmK genes. The translation product of vlmJ exhibits similarity to diacylglycerol kinases, while the translation product of vlmK exhibits a weak similarity to the MmgE/PrpD superfamily of proteins. This superfamily includes 2-methylcitrate dehydratase. This communication describes the isolation and structure elucidation of valanimycin hydrate from vlmJ and vlmK mutants of S. viridifaciens. Additional studies have shown that the conversion of valanimycin hydrate into valanimycin by S. viridifaciens requires both the vlmJ and vlmK genes and that VlmJ catalyzes the ATP-dependent phosphorylation of the hydroxyl group of valanimycin hydrate prior to VlmK-catalyzed dehydration.

New prodiginines from a ketosynthase swap.

The prodiginine antibiotics exhibit antitumor and immunosuppressive activity. In this issue of Chemistry & Biology, Reynolds and coworkers demonstrate that new prodiginines can be obtained by substituting a FabH ketosynthase for the RedP ketosynthase in the undecylprodiginine biosynthetic gene cluster.

Draft Genome Sequence of Streptomyces vitaminophilus ATCC 31673, a Producer of Pyrrolomycin Antibiotics, Some of Which Contain a Nitro Group.

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. The 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content.

Valanimycin biosynthesis: investigations of the mechanism of isobutylhydroxylamine incorporation.

[reaction: see text] The incorporation of [(15)N, (18)O]-isobutylhydroxylamine into the antibiotic valanimycin by Streptomyces viridifaciens has been shown to proceed with loss of the (18)O label, thereby demonstrating that the azoxy oxygen atom of valanimycin is not derived from the oxygen atom of isobutylhydroxylamine.

Biosynthetic investigations of lactonamycin and lactonamycin z: cloning of the biosynthetic gene clusters and discovery of an unusual starter unit.

The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes coding for the biosynthesis of l-rhodinose, the sugar found in lactonamycin, and genes similar to those in the tetracenomycin biosynthetic gene cluster. Since lactonamycin production by S. rishiriensis could not be sustained, additional proof for the identity of the S. rishiriensis cluster was obtained by cloning the lactonamycin Z gene cluster from Streptomyces sanglieri. Partial sequencing of the S. sanglieri cluster revealed 15 genes that exhibited a very high degree of similarity to genes within the lactonamycin cluster, as well as an identical organization. Double-crossover disruption of one gene in the S. sanglieri cluster abolished lactonamycin Z production, and production was restored by complementation. These results confirm the identity of the genetic locus cloned from S. sanglieri and indicate that the highly similar locus in S. rishiriensis encodes lactonamycin biosynthetic genes. Precursor incorporation experiments with S. sanglieri revealed that lactonamycinone is biosynthesized in an unusual manner whereby glycine or a glycine derivative serves as a starter unit that is extended by nine acetate units. Analysis of the gene clusters and of the precursor incorporation data suggested a hypothetical scheme for lactonamycinone biosynthesis.

Cloning and characterization of the pyrrolomycin biosynthetic gene clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. strain UC 11065.

The pyrrolomycins are a family of polyketide antibiotics, some of which contain a nitro group. To gain insight into the nitration mechanism associated with the formation of these antibiotics, the pyrrolomycin biosynthetic gene cluster from Actinosporangium vitaminophilum was cloned. Sequencing of ca. 56 kb of A. vitaminophilum DNA revealed 35 open reading frames (ORFs). Sequence analysis revealed a clear relationship between some of these ORFs and the biosynthetic gene cluster for pyoluteorin, a structurally related antibiotic. Since a gene transfer system could not be devised for A. vitaminophilum, additional proof for the identity of the cloned gene cluster was sought by cloning the pyrrolomycin gene cluster from Streptomyces sp. strain UC 11065, a transformable pyrrolomycin producer. Sequencing of ca. 26 kb of UC 11065 DNA revealed the presence of 17 ORFs, 15 of which exhibit strong similarity to ORFs in the A. vitaminophilum cluster as well as a nearly identical organization. Single-crossover disruption of two genes in the UC 11065 cluster abolished pyrrolomycin production in both cases. These results confirm that the genetic locus cloned from UC 11065 is essential for pyrrolomycin production, and they also confirm that the highly similar locus in A. vitaminophilum encodes pyrrolomycin biosynthetic genes. Sequence analysis revealed that both clusters contain genes encoding the two components of an assimilatory nitrate reductase. This finding suggests that nitrite is required for the formation of the nitrated pyrrolomycins. However, sequence analysis did not provide additional insights into the nitration process, suggesting the operation of a novel nitration mechanism.

Characterization of a nonspecific phosphopantetheinyl transferase from Pseudomonas syringae pv. syringae FF5.

The 4'-phosphopantetheinyl transferases (PPTases) catalyze the transfer of a 4'-phosphopantetheine moiety from coenzyme A to phosphopantetheine-dependent carrier proteins. The carrier proteins (CPs) are required for the biosynthesis of peptides synthesized by nonribosomal peptide synthases and the biosynthesis of fatty acids and polyketides. A single PPTase (PcpS) is present in the pathogenic bacterium Pseudomonas aeruginosa. Several pathovars of Pseudomonas syringae produce the chlorosis-inducing phytotoxin coronatine. Structural genes for coronatine biosynthesis include two ACPs, two ACP domains, and one peptidyl carrier protein (PCP) domain. To gain insight into factors affecting coronatine biosynthesis, the PPTase of P. syringae pv. syringae FF5 has been investigated. A single PPTase gene (pspT) was amplified from this organism by PCR. The translation product PspT exhibited 62% identity to PcpS as well as higher levels of identity to other, uncharacterized Pseudomonad PPTases. PspT was overproduced in soluble form in Escherichia coli and its enzymatic properties were compared with those of PcpS. PspT exhibited broad substrate specificity, and it displayed the highest activity with a PCP domain. In contrast, the most efficient substrates for PcpS are CPs from primary metabolism. These results indicate phosphopantetheinyl transferases from different Pseudomonas sp. may vary significantly in their enzymatic properties.

Biochemical characterization of VlmL, a Seryl-tRNA synthetase encoded by the valanimycin biosynthetic gene cluster.

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

A novel valanimycin-resistance determinant (vlmF) from Streptomyces viridifaciens MG456-hF10.

A novel valanimycin-resistance determinant (vImF) was isolated from a cosmid containing Streptomyces viridifaciens DNA that leads to valanimycin production in Streptomyces lividans. Expression of the vImF gene in both Escherichia coli and S. lividans provided valanimycin resistance. The nucleotide sequence of vImF consists of 1206 bp and the deduced amino acid sequence encodes a polypeptide with 12 putative transmembrane-spanning segments and a calculated pI of 10.1. VImF shows significant similarities to other known or putative transmembrane efflux proteins that confer antibiotic resistance, but it appears to be specific for valanimycin. The sequence similarities suggest that VImF is a member of the DHA12 family within the major facilitator superfamily of transport proteins and that it is probably involved in active valanimycin efflux energized by a proton-dependent electrochemical gradient.

Characterization of Cfa1, a monofunctional acyl carrier protein involved in the biosynthesis of the phytotoxin coronatine.

Cfa1 was overproduced in Escherichia coli and Pseudomonas syringae, and the degree of 4'-phosphopantetheinylation was determined. The malonyl-coenzyme A:acyl carrier protein transacylase (FabD) of P. syringae was overproduced and shown to catalyze malonylation of Cfa1, suggesting that FabD plays a role in coronatine biosynthesis. Highly purified Cfa1 did not exhibit self-malonylation activity.

Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine.

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.

Regioselective nitration of tryptophan by a complex between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase.

Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79-82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H(4)B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position.