Identification of the Azaserine Biosynthetic Gene Cluster Implicates Hydrazine as an Intermediate to the Diazo Moiety.

Azaserine (1) is a natural product and nonproteinogenic amino acid containing a diazo group. Here we report the biosynthetic gene cluster for 1 from Glycomyces harbinensis. We then use isotopic feeding, gene deletion, and biochemical experiments to support a pathway whereby hydrazinoacetic acid (2) and a peptidyl carrier protein-loaded serine (3) are intermediates on route to the final natural product 1.

Free Piperazic Acid as a Precursor to Nonribosomal Peptides.

Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.

Natural Products Produced in Culture by Biosynthetically Talented Salinispora arenicola Strains Isolated from Northeastern and South Pacific Marine Sediments.

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.

Synthetic and biosynthetic routes to nitrogen-nitrogen bonds.

The nitrogen-nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles. Such functional groups are found in >300 known natural products. Such N-N bond-containing functional groups are also found in significant percentage of clinical drugs. Therefore, there is wide interest in synthetic and enzymatic methods to form nitrogen-nitrogen bonds. In this review, we summarize synthetic and biosynthetic approaches to diverse nitrogen-nitrogen-bond-containing functional groups, with a focus on biosynthetic pathways and enzymes.

Biosynthesis of the N-N-Bond-Containing Compound l-Alanosine.

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

Functional analysis of methyltransferases participating in streptothricin-related antibiotic biosynthesis.

Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated d-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the l-ß-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N'-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic.

UNLABELLED: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ß-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. IMPORTANCE: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than ß-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP-[(32)P]-PPi exchange assays with the detection of (32)P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a yellow 18-molybdopyrophosphate anion ([(P2O7)Mo18O54](4-)). [(P2O7)Mo18O54](4-) was further reduced by ascorbic acid to give a more readily distinguishable blue coloration. Here we demonstrate the lab protocols for the colorimetric assay of PPi released in A-domain reactions.