Ronald J. Parry

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.

AN NADPH:FAD OXIDOREDUCTASE FROM THE VALANIMYCIN PRODUCER, STREPTOMYCES VIRIDIFACIENS : CLONING, ANALYSIS, AND OVEREXPRESSION

The valanimycin producer Streptomyces viridifaciens contains a two-component enzyme system that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine. One component of this enzyme system is isobutylamine hydroxylase, and the other component is a flavin reductase. The gene (vlmR) encoding the flavin reductase required by isobutylamine hydroxylase has been cloned from S. viridifaciens by chromosome walking. The gene codes for a protein of 194 amino acids with a calculated mass of 21,265 Da and a calculated pI of 10.2. Overexpression of thevlmR gene in Escherichia coli as an N-terminal His-tag derivative yielded a soluble protein that was purified to homogeneity. Removal of the N-terminal His-tag from the overexpressed protein by thrombin cleavage also produced a soluble protein. Both forms of the protein exhibited a high degree of flavin reductase activity, and the thrombin-cleaved form functioned in combination with isobutylamine hydroxylase to catalyze the conversion of isobutylamine to isobutylhydroxylamine. Kinetic data indicate that the overexpressed protein utilizes FAD and NADPH in preference to FMN, riboflavin, and NADH. The deduced amino acid sequence of the VlmR protein exhibited similarity to several other flavin reductases that may constitute a new family of flavin reductases.

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.

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 (H4B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H4B. 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.

Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin.

Streptomyces viridifaciens MG456‐hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Strepto‐myces lividans TK24. Subcloning of DNA from the valanimycin‐producing cosmid has led to the identi‐fication of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar‐boxylase gene ( vlmD ), a valanimycin resistance gene ( vlmF ), at least two regulatory genes ( vlmE , vlmI ), two genes encoding a flavin monooxygenase ( vlmH , vlmR ), a seryl tRNA synthetase gene ( vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of l ‐valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5′ end.

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.

Biosynthesis of aristeromycln: Evidence for the intermediacy of a 4β-hydroxymethyl-1α, 2α, 3α-trihydroxycyclopentanetriol.

Abstract Evidence for the intermediacy of a 4β-hydroxymethyl-1α, 2α, 3α-trihydroxycyclopentanetdol ( 5 or 6 ) in the biosynthesis of the nucleoside antibiotic aristeromycin ( 1 ) has been obtained by administration of doubly-labeled forms of D-glucose to the fermentation broth of Streptomyces citricolor followed by trapping of the tetrol 5 using isotope dilution methods.

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.

Investigations of coronatine biosynthesis. Overexpression and assay of CmaT, a thioesterase involved in coronamic acid biosynthesis

Abstract The protein (CmaT) encoded by the cmaT gene of the coronamic acid biosynthetic gene cluster has been overexpressed in Escherichia coli in soluble and active form fused to the carboxyl terminus of MalE, the maltose-binding protein. CmaT was also overexpressed in E. coli as an N-terminal His-tagged protein. The N-terminal His-tagged form of CmaT was produced in insoluble form, but it could be refolded to obtain CmaT in soluble and highly active form. Both the MalE-CmaT fusion protein and the refolded His-tagged CmaT protein exhibited esterase activity.

Determination of the absolute configuration of (−)-S-(2-carboxypropyl)-L-cysteine

Abstract The absolute configuration of the naturally occurring amino acid (−)-S-(2-carboxypropyl)-L-cysteine has been determined by asymmetric synthesis.