Biosynthesis of Hexahydroxyperylenequinone Melanin via Oxidative Aryl Coupling by Cytochrome P-450 in Streptomyces griseus

ABSTRACT Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NADP+ reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter.

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Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase

Nature Chemical Biology ◽

10.1038/nchembio811 ◽

2006 ◽

Vol 2 (9) ◽

pp. 494-502 ◽

Cited By ~ 86

Author(s):

Michael B Austin ◽

Tamao Saito ◽

Marianne E Bowman ◽

Stephen Haydock ◽

Atsushi Kato ◽

...

Keyword(s):

Fatty Acid ◽

Dictyostelium Discoideum ◽

Polyketide Synthase ◽

Type I ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Hybrid Type ◽

Acid Type ◽

Fatty Acid Type

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Bacterial Enzymes Catalyzing the Synthesis of 1,8-Dihydroxynaphthalene, a Key Precursor of Dihydroxynaphthalene Melanin, fromSorangium cellulosum

Applied and Environmental Microbiology ◽

10.1128/aem.00258-18 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 8

Author(s):

Yusuke Sone ◽

Shuto Nakamura ◽

Makoto Sasaki ◽

Fumihito Hasebe ◽

Seung-Young Kim ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Optical Purity ◽

Brown Pigment ◽

Type I ◽

Enzymatic System ◽

Type Iii Polyketide Synthase ◽

Content Type ◽

Type Iii ◽

Bacterial Origin

ABSTRACT1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting ofsoceCHS1,bdsA, andbdsBfrom the Gram-negative bacteriumSorangium cellulosum. Heterologous expression ofsoceCHS1,bdsA, andbdsBinStreptomyces coelicolorcaused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN)in vitro. T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on thesi-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCEAlthough the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.

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Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00407-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4916-4923 ◽

Cited By ~ 41

Author(s):

Chiaki Nakano ◽

Hiroki Ozawa ◽

Genki Akanuma ◽

Nobutaka Funa ◽

Sueharu Horinouchi

Keyword(s):

Bacillus Subtilis ◽

Polyketide Synthase ◽

Bacterial Genome ◽

Fatty Acyl ◽

Gram Positive ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Vitro Analysis

ABSTRACT Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.

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A Type I/Type III Polyketide Synthase Hybrid Biosynthetic Pathway for the Structurally UniqueansaCompound Kendomycin

ChemBioChem ◽

10.1002/cbic.200800456 ◽

2008 ◽

Vol 9 (16) ◽

pp. 2711-2721 ◽

Cited By ~ 40

Author(s):

Silke C. Wenzel ◽

Helge B. Bode ◽

Irene Kochems ◽

Rolf Müller

Keyword(s):

Biosynthetic Pathway ◽

Polyketide Synthase ◽

Type I ◽

Type Iii Polyketide Synthase ◽

Type Iii

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In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

Chemical Science ◽

10.1039/c5sc01013f ◽

2015 ◽

Vol 6 (8) ◽

pp. 5076-5085 ◽

Cited By ~ 25

Author(s):

H. Sucipto ◽

J. H. Sahner ◽

E. Prusov ◽

S. C. Wenzel ◽

R. W. Hartmann ◽

...

Keyword(s):

Natural Products ◽

Polyketide Synthase ◽

Ring Formation ◽

Type I ◽

Pyrone Ring ◽

In Vitro Reconstitution

α-Pyrone rings exist in many polyketide synthase (PKS) derived natural products. We report the first in vitro reconstitution of α-pyrone ring formation by a type I PKS using chemically synthesized substrates.

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Molecular Characterization and Differential Expression of an Aromatic Heptaketide Producing Type III Plant Polyketide Synthase From Himalayan Rhubarb

10.21203/rs.3.rs-542003/v1 ◽

2021 ◽

Author(s):

Shahzad A. Pandith ◽

Niha Dhar ◽

Sumedha Bhosale ◽

Vitthal T. Barvkar ◽

Sumeer Razdan ◽

...

Keyword(s):

Polyketide Synthase ◽

Uv Light ◽

Phylogenetic Analyses ◽

Transcript Abundance ◽

Single Copy ◽

Medical Systems ◽

Regeneration System ◽

Multiple Sequence ◽

Type Iii

Abstract Rheum australe (Himalayan Rhubarb, Polygonaceae) is an endangered medicinal and vegetable herb with known efficacy in different traditional medical systems. The age-old remedying properties of this perennial species are ascribed to the bio-active phytoconstituents viz anthraquinones, stilbenoids, chromones and dietary flavonoids. These metabolites share a major contribution of their synthesis from polyketide pathway which primarily involves the intricate Type III polyketide synthases (PKSs). In this context, present study was focussed on characterization of an important PKS member aloesone synthase (RaALS) known to catalyze six polyketide extensions of the starter acetyl CoA to generate aloesone. A full-length cDNA (1176 bp, ~ 42 kDa) was cloned and heterologously expressed in microbial hosts. Multiple sequence alignment and phylogenetic analyses revealed that RaALS shares high degree of similarity with orthologous PKSs. Model validation and donor-acceptor interactions were assessed using homology modeling and molecular docking studies. Further, southern blotting determined the existence of RaALS as single copy gene. qRT-PCR analyses revealed that higher expression of RaALS in leaves followed by stem and root corroborated well with the metabolite accumulation. Its expression was also found to be regulated by altitude. Additionally, an in vitro regeneration system was developed to evaluate the effect of various abiotic stressors viz methyl jasmonate, salicylic acid and UV light on the transcript abundance of RaALS vis-à-vis identified putative cis-regulatory promoter elements using genome walking approach. The study would act as a prelude to utilize this medical herb for prospective metabolic engineering attempts aimed at augmenting bio-active metabolite production for commercial purposes.

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Faculty Opinions recommendation of Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1040317.489268 ◽

2006 ◽

Author(s):

David Newman

Keyword(s):

Fatty Acid ◽

Dictyostelium Discoideum ◽

Polyketide Synthase ◽

Type I ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Hybrid Type ◽

Acid Type ◽

Fatty Acid Type

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Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj20020953 ◽

2002 ◽

Vol 367 (3) ◽

pp. 781-789 ◽

Cited By ~ 31

Author(s):

Nobutaka FUNA ◽

Yasuo OHNISHI ◽

Yutaka EBIZUKA ◽

Sueharu HORINOUCHI

Keyword(s):

Amino Acid ◽

Active Site ◽

Polyketide Synthase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Type Iii Polyketide Synthase ◽

Melanin Biosynthesis ◽

Type Iii ◽

Malonyl Coa

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys138, His270 and Asn303 of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr224 is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr224 by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala305 mutants and identification of their products showed that the substitution of Ala305 by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr224 (important for starter substrate selection) and Ala305 (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

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