In vitro reconstitution of the catabolic reactions catalyzed by PcaHG, PcaB, and PcaL: the protocatechuate branch of the beta-ketoadipate pathway in Rhodococcus jostii RHA1

The ß-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the ß-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the ß-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, ß-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, y-carboxymuconolactone, muconolactone, and ß-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of y-carboxy-muconolactone decarboxylase and ß-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of ß-ketoadipate pathways deposited in the genetic databas

Synthesis of Unnatural Flavonoids and Stilbenes by Exploiting the Plant Biosynthetic Pathway in Escherichia coli

SummaryFlavonoids and stilbenes have attracted much attention as potential targets for nutraceuticals, cosmetics, and pharmaceuticals. We have developed a system for producing “unnatural” flavonoids and stilbenes in Escherichia coli. The artificial biosynthetic pathway included three steps. These included a substrate synthesis step for CoA esters synthesis from carboxylic acids by 4-coumarate:CoA ligase, a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthase and chalcone isomerase, and into stilbenes by stilbene synthase, and a modification step for modification of the flavanones by flavone synthase, flavanone 3β-hydroxylase and flavonol synthase. Incubation of the recombinant E. coli with exogenously supplied carboxylic acids led to production of 87 polyketides, including 36 unnatural flavonoids and stilbenes. This system is promising for construction of a larger library by employing other polyketide synthases and modification enzymes

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

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

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

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

<i>In vitro</i> reconstitution of the catabolic reactions catalyzed by PcaHG, PcaB, and PcaL: the protocatechuate branch of the β-ketoadipate pathway in <i>Rhodococcus jostii</i> RHA1

<div><p>The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of <i>Rhodococcus jostii</i> was reconstituted <i>in vitro</i>. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-<i>cis</i>,<i>cis</i>-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the <i>in vitro</i> reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.</p></div

Fosfomycin Biosynthesis <i>via</i> Transient Cytidylylation of 2‑Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, <i>etc</i>. Its biosynthesis starts with the formation of a carbon–phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 <i>N</i>-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 <i>C</i>-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism <i>via</i> such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family

Curcuminoid Biosynthesis by Two Type III Polyketide Synthases in the Herb Curcuma longa*S⃞

Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III polyketide synthases. One of the type III polyketide synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold