A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis

Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here we report a route to incorporate phenylglycine residues in peptide thioesters without significant racemisation.

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Glycopeptide antibiotic biosynthesis: Enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.221582098 ◽

2001 ◽

Vol 98 (26) ◽

pp. 14901-14906 ◽

Cited By ~ 73

Author(s):

H. Chen ◽

C. C. Tseng ◽

B. K. Hubbard ◽

C. T. Walsh

Keyword(s):

Amino Acid ◽

Antibiotic Biosynthesis ◽

Glycopeptide Antibiotic

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Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor

mBio ◽

10.1128/mbio.00768-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Inna Zukher ◽

Michael Pavlov ◽

Darya Tsibulskaya ◽

Alexey Kulikovsky ◽

Tatyana Zyubko ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Antibiotic Production ◽

Cell Uptake ◽

Antibiotic Biosynthesis ◽

Formyl Group ◽

Content Type ◽

E Coli ◽

Precursor Peptide ◽

Peptide Length

ABSTRACT Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. coli mcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug. IMPORTANCE Escherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.

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Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part I. The synthesis of echinomycin

Canadian Journal of Microbiology ◽

10.1139/m84-111 ◽

1984 ◽

Vol 30 (6) ◽

pp. 721-729 ◽

Cited By ~ 3

Author(s):

D. Gauvreau ◽

M. J. Waring

Keyword(s):

Amino Acid ◽

Stationary Phase ◽

Glass Wool ◽

Antibiotic Biosynthesis ◽

Resting Cells ◽

Intact Cells ◽

Significant Contamination ◽

Seed Medium ◽

Putative Precursor ◽

Kinetics Of

Washed suspensions of Streptomyces echinatus, and protoplasts derived from them, have been shown to synthesise echinomycin in the absence of growth. Protoplast suspensions free from significant contamination with unlysed mycelia are obtained by incubation with lysozyme followed by filtration through layers of tightly packed glass wool. Although physiologically young cells produce a better yield of protoplasts, optimal antibiotic biosynthesis is achieved with protoplasts prepared from mycelia about to enter the stationary phase of growth, i.e., approximately 24 h after inoculation into a nutrient broth – salts seed medium. As judged by the incorporation of label from L-[methyl-14C]methionine, echinomycin synthesis proceeds for about 1 h after preparation of washed suspensions, but the kinetics of incorporation by intact cells and protoplasts are different. Uptake of labelled methionine by protoplasts is critically dependent upon the presence of sucrose as osmotic stabiliser and is drastically reduced if galactose, calcium, or magnesium is omitted from the suspending buffer. Uptake by intact, washed cells is essentially independent of nutrients in the medium. Small quantities of 11 materials other than echinomycin are detectable in chloroform extracts after labelling with L-[methyl-14C]methionine; some of these may represent precursors in the biosynthesis of the antibiotic. All amino acid constituents of echinomycin as well as tryptophan, a putative precursor of the quinoxaline chromophores, are actively incorporated into echinomycin by protoplasts and resting cells, but not with equal efficiency.

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3-Amino-5-Hydroxybenzoic Acid in Antibiotic Biosynthesis. IX. The Status of Reduced Derivatives in Mitomycin Biosynthesis

Australian Journal of Chemistry ◽

10.1071/ch9871011 ◽

1987 ◽

Vol 40 (5) ◽

pp. 1011 ◽

Cited By ~ 1

Author(s):

RW Rickards ◽

V Rukachaisirikul

Keyword(s):

Amino Acid ◽

Isotopic Composition ◽

Antibiotic Biosynthesis ◽

Hydroxybenzoic Acid ◽

The Status ◽

Metabolic Precursors ◽

Hydroxybenzyl Alcohol

The addition of (methylene-2H2)-3-amino-5-hydroxybenzyl alcohol (8) hydrochloride to growing cultures of Streptomyces verticillatus inhibits production of the mitomycin antibiotic porfiromycin (2) by the microorganism. No incorporation of deuterium into porfiromycin occurs, and the isotopic composition of the alcohol recovered from the fermentation is unchanged. The significance of these results with regard to the immediate metabolic precursors of 3-amino-5-hydroxybenzoic acid (3) and the reduction stages involved in the established conversion of this amino acid into mitomycins are discussed.

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The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus

Microbiology ◽

10.1099/mic.0.29043-0 ◽

2006 ◽

Vol 152 (10) ◽

pp. 2969-2983 ◽

Cited By ~ 74

Author(s):

Xihou Yin ◽

T. Mark Zabriskie

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gene Cluster ◽

Genetic Locus ◽

Biosynthetic Gene Cluster ◽

Antibiotic Biosynthesis ◽

Peptide Antibiotic ◽

Amino Acid Residues ◽

Biosynthetic Gene ◽

Condensation Domain

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr8, is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.

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Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

Journal of Bacteriology ◽

10.1128/jb.00045-15 ◽

2015 ◽

Vol 197 (12) ◽

pp. 2062-2071 ◽

Cited By ~ 6

Author(s):

Yawei Zhao ◽

Rongrong Feng ◽

Guosong Zheng ◽

Jinzhong Tian ◽

Lijun Ruan ◽

...

Keyword(s):

Amino Acid ◽

Metabolic Flux ◽

Phenylacetic Acid ◽

Antibiotic Biosynthesis ◽

Catabolic Pathway ◽

Positive Role ◽

Content Type ◽

Streptomyces Pristinaespiralis ◽

Amino Acid Precursors ◽

Tetr Family Regulator

ABSTRACTPristinamycin I (PI), produced byStreptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded bySSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, fromCorynebacterium glutamicumserves as a transcriptional repressor of thepaagenes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion ofpaaRled to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue fromC. glutamicum, PaaR is also involved in the suppression ofpaaexpression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway ofl-phenylglycine (l-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription ofpaagenes in a ΔpaaRmutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux towardl-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ΔpaaRmutant was restored byl-Phg supplementation as well as by deletion of thepaaABCDEoperon in the ΔpaaRmutant. Altogether, this study provides new insights into the regulation of PI biosynthesis byS. pristinaespiralis.IMPORTANCEA better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues forStreptomycesstrain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription ofpaagenes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors,l-phenylglycine, inStreptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis inStreptomycesstrains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.

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The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142372 ◽

1986 ◽

Vol 44 ◽

pp. 150-151

Author(s):

M.K. Lamvik ◽

L.L. Klatt

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Staining Pattern ◽

Banding Patterns ◽

Mass Thickness ◽

New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

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