Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α‐Hydroxylated Products from C‐19 Steroids

Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high‐throughput screening of a compound library (ca. 17 000 ligands). Structural analogues of the type I hits were searched for biotechnologically relevant compounds, and this led us to select C‐19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli‐based whole‐cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11‐oxoandrostenedione. A detailed ¹H and ¹³C NMR characterization of the product(s) from C‐19 steroids revealed that CYP260A1 is the very first 1α‐steroid hydroxylase.     

New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non‐activated C?H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)‐nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.     

A Membrane‐Bound Prenyltransferase Catalyzes the O‐Prenylation of 1,6‐Dihydroxyphenazine in the Marine Bacterium Streptomyces sp. CNQ‐509

Streptomyces sp. CNQ‐509 produces the rare O‐prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O‐prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ‐509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine‐1‐carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6‐dihydroxyphenazine geranyltransferase, which catalyzes the C?O bond formation between C‐1 of the geranyl moiety and O‐6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O‐prenyl transfer to a phenazine.     

Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307‐9

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl‐tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307–9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase–nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly‐line oxidative modifications.     

Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.     

Epimerization at C‐3′′ in Butirosin Biosynthesis by an NAD+‐Dependent Dehydrogenase BtrE and an NADPH‐Dependent Reductase BtrF

Butirosin is an aminoglycoside antibiotic consisting two epimers at C‐3′′ of ribostamycin/xylostasin with a unique 4‐amino‐2‐hydroxybutyrate moiety at C‐1 of the aminocyclitol 2‐deoxystreptamine (2DOS). To date, most of the enzymes encoded in the biosynthetic gene cluster for butirosin, from the producing strain Bacillus circulans, have been characterized. A few unknown functional proteins, including nicotinamide adenine dinucleotide cofactor‐dependent dehydrogenase/reductase (BtrE and BtrF), are supposed to be involved in the epimerization at C‐3′′ of butirosin B/ribostamycin but remain to be characterized. Herein, the conversion of ribostamycin to xylsostasin by BtrE and BtrF in the presence of NAD⁺ and NADPH was demonstrated. BtrE oxidized the C‐3′′ of ribostamycin with NAD⁺ to yield 3′′‐oxoribostamycin. BtrF then reduced the generated 3′′‐oxoribostamycin with NADPH to produce xylostasin. This reaction step was the last piece of butirosin biosynthesis to be described.     

Random copolymerization of 2,2‐dimethyltri methylene carbonate and ε‐caprolactone using a rare‐earth tris(4‐tert‐butylphenolate)s as a single‐component initiator

Highly random copolymers of 2,2‐dimethyltrimethylene carbonate (DTC) and ε‐caprolactone (CL) were synthesized by single component rare‐earth tris(4‐tert‐butylphenolate)s [Ln(OTBP)₃] for the first time. The influences of reaction conditions on the copolymerization initiated by La(OTBP)₃ have been examined in detail. The monomer reactivity ratios of DTC and CL determined by the Fineman–Ross method are 4.0 for r_DTC and 0.27 for r_CL. The microstructure of the copolymer was determined by the analyses of the diads DTC–DTC, DTC–CL, CL–DTC and CL–CL of the ¹H NMR spectra. The high degree of randomness of the chain structure was further confirmed by the ¹³C NMR spectra and differential scanning calorimetry. The thermal properties of the copolymers as a function of composition are reported. The mechanism investigated by ¹H NMR data indicates that the rare‐earth tris(4‐tert‐butylphenolate)s initiate the ring‐opening copolymerization of DTC and CL with acyl‐oxygen bond cleavages of the monomers. Copyright © 2004 Society of Chemical Industry     

5‐Benzylidenethiazolidin‐4‐ones as Multitarget Inhibitors of Bacterial Mur Ligases

Mur ligases participate in the intracellular path of bacterial peptidoglycan biosynthesis and constitute attractive, although so far underexploited, targets for antibacterial drug discovery. A series of hydroxy‐substituted 5‐benzylidenethiazolidin‐4‐ones were synthesized and tested as inhibitors of Mur ligases. The most potent compound 5 a was active against MurD–F with IC₅₀ values between 2 and 6 μm, making it a promising multitarget inhibitor of Mur ligases. Antibacterial activity against different strains, inhibitory activity against protein kinases, mutagenicity and genotoxicity of 5 a were also investigated, and kinetic and NMR studies were conducted.     

A Unique Amino Transfer Mechanism for Constructing the β‐Amino Fatty Acid Starter Unit in the Biosynthesis of the Macrolactam Antibiotic Cremimycin

Cremimycin is a 19‐membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β‐amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β‐amino‐acid‐transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3‐aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin‐type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β‐position of a non‐2‐enoic acid thioester, followed by hydrolysis of the thioester to give N‐carboxymethyl‐3‐aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD‐dependent glycine oxidase homologue, CmiS2, to produce 3‐aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β‐amino acid biosynthesis.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Synthesis and spectroelectrochemistry of dithieno(3,2‐b:2′,3′‐d)pyrrole derivatives

New π‐conjugated polymers containing dithieno(3,2‐b:2′,3′‐d)pyrrole (DTP) were successfully synthesized via electropolymerization. The effect of structural differences on the electrochemical and optoelectronic properties of the 4‐[4H‐dithieno(3,2‐b:2′,3′‐d)pyrrol‐4‐yl]aniline (DTP–aryl–NH₂), 10‐[4H‐dithiyeno(3,2‐b:2′,3′‐d)pirol‐4‐il]dekan‐1‐amine (DTP–alkyl–NH₂), and 1,10‐bis[4H‐dithieno(3,2‐b:2′,3′‐d)pyrrol‐4‐yl] decane (DTP–alkyl–DTP) were investigated. The corresponding polymers were characterized by cyclic voltammetry, NMR (¹H‐NMR and ¹³C‐NMR), and ultraviolet–visible spectroscopy. Changes in the electronic nature of the functional groups led to variations in the electrochemical properties of the π‐conjugated systems. The electroactive polymer films revealed redox couples and exhibited electrochromic behavior. The replacement of the DTP–alkyl–DTP unit with DTP–aryl–NH₂ and DTP–alkyl–NH₂ resulted in a lower oxidation potential. Both the poly(10‐(4H‐Dithiyeno[3,2‐b:2′,3′‐d]pirol‐4‐il)dekan‐1‐amin) (poly(DTP–alkyl–NH₂)) and poly(1,10‐bis(4H‐dithieno[3,2‐b:2′,3′‐d]pyrrol‐4‐yl) decane) (poly(DTP–alkyl–DTP)) films showed multicolor electrochromism and also fast switching times (<1 s) in the visible and near infrared regions. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40701.     

Yeast‐mediated enantioselective synthesis of chiral R‐(+)‐ and S‐(−)‐1‐phenyl‐1‐butanol from prochiral phenyl n‐propyl ketone in hexane–water biphasic culture

BACKGROUND: The hydrophobic phenyl n‐propyl ketone was used as a model compound to examine alcohol dehydrogenase activity in Saccharomyces cerevisiae mediated cell culture. Parameters such as pH, hexane‐to‐water volume percentage, and the amount of cofactor Zn²⁺ ion for either cell growth or reduction were studied to see their effect on the enantioselectivity toward the product R‐(+)‐ or S‐(?)‐1‐phenyl‐1‐butanol. RESULTS: The pH for cell growth in aqueous culture was 7.0, while the pH for reduction in the aqueous portion of the biphasic culture was 5.0. Without Zn²⁺ ion the biphasic cultures of middle to high hexane‐to‐water volume percentage exhibited an R‐(+)‐1‐phenyl‐1‐butanol enantiomeric excess of 53.7% to > 99%. Without Zn²⁺ ion the biphasic cultures at low hexane‐to‐water volume percentage possessed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 14.5–46.5%. Exclusively, the enantioselectivity for biphasic cultures containing Zn²⁺ ion was an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 27.5% to > 99%. Reduction mediated in aqueous culture with varied amount of Zn²⁺ ion by the yeast Candida utilis also showed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 79.2–95.4%. CONCLUSION: The enantioselectivity of S. cerevisiae mediated biphasic culture reduction of phenyl n‐propyl ketone can be manipulated through the cofactor Zn²⁺ ion and the hexane volume percentage of the biphasic culture. Copyright © 2008 Society of Chemical Industry     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Expanding the Binding Envelope of CYP51 Inhibitors Targeting Trypanosoma cruzi with 4‐Aminopyridyl‐Based Sulfonamide Derivatives

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4‐aminopyridyl‐based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub‐nanomolar compound 3 , have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X‐ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground‐state conformation of CYP51 drug–target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.     

Synthesis and characterization of new cardo poly(bisbenzothiazole)s from 1,1‐bis(4‐amino‐3‐mercaptophenyl)‐4‐tert‐butylcyclohexane dihydrochloride

A new monomer 1,1‐bis(4‐amino‐3‐mercaptophenyl)‐4‐tert‐butylcyclohexane dihydrochloride, bearing the bulky pendant 4‐tert‐butylcyclohexylidene group, was synthesized from 4‐tert‐butylcyclohexanone in three steps. Its chemical structure was characterized by ¹H NMR, ¹³C NMR, MS, FTIR, and EA. Aromatic poly(bisbenzothiazole)s (PBTs V) were prepared from the new monomer and five aromatic dicarboxylic acids by direct polycondensation. The inherent viscosities were in the range of 0.63–2.17 dL/g. These polymers exhibited good solubility and thermal stability. Most of the prepared PBTs V were soluble in various polar solvents. Thermogravimetric analysis showed the decomposition temperatures at 10% weight loss that were in the range of 495–534°C in nitrogen. All the PBTs V, characterized by X‐ray diffraction, were amorphous. The UV absorption spectra of PBTs V showed a range of λ_max from 334 to 394 nm. All the PBTs V prepared had evident fluorescence emission peaks, ranging from 423 to 475 nm with different intensity. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 2000–2008, 2006     

Synthesis,characterization, and antimicrobial properties of oligo‐4‐[(pyridine‐3‐yl‐methylene) amino] phenol

The oxidative polycondensation reaction conditions of 4‐[(pyridine‐3‐yl‐methylene) amino]phenol (4‐PMAP) were studied using H₂O₂, atmospheric O₂, and NaOCl oxidants in an aqueous alkaline medium between 30°C and 90°C. Synthesized oligo‐4‐[(pyridine‐3‐yl‐methylene) amino] phenol (O‐4‐PMAP) was characterized by ¹H‐, ¹³C NMR, FTIR, UV–vis, size exclusion chromatography (SEC), and elemental analysis techniques. The yield of O‐4‐PMAP was found to be 32% (for H₂O₂ oxidant), 68% (for atmospheric O₂ oxidant), and 82% (for NaOCl oxidant). According to the SEC analysis, the number–average molecular weight, weight–average molecular weight, and polydispersity index values of O‐4‐PMAP was found to be 5767, 6646 g mol^?1, and 1.152, respectively, using H₂O₂, and 4540, 5139 g mol^?1, and 1.132, respectively, using atmospheric O₂, and 9037, 9235 g mol^?1, and 1.022, using NaOCl, respectively. According to TG and DSC analyses, O‐4‐PMAP was more stable than 4‐PMAP against thermal decomposition. The weight loss of O‐4‐PMAP was found to be 94.80% at 1000°C. Also, antimicrobial activities of the oligomer were tested against B. cereus, L. monocytogenes, B. megaterium, B. subtilis, E. coli, Str. thermophilus, M. smegmatis, B. brevis, E. aeroginesa, P. vulgaris, M. luteus, S. aureus, and B. jeoreseens. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3327–3333, 2006     

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.     

Poly‐α,β‐(3‐hydroxypropyl)‐DL‐aspartamide: A new drug carrier

Poly‐α,β‐(3‐hydroxypropyl)‐DL ‐aspartamide (PHPA) was synthesized by the ring‐open reaction of polysuccinimide (PSI) and 3‐hydroxypropylamine. The polymer was characterized by ¹H‐NMR, ¹³C‐NMR, FTIR, and GPC. Mark–Houwink coefficients were obtained from viscometry and GPC measurements, K = 5.53 × 10⁻³ and α = 0.78 in water. The acute toxicity of PHPA was examined and it revealed no death in ICR mice up to the dose treated of 15.3 kg/kg, and hematological parameters showed no significant difference between treated and control animals. The potential use of PHPA as a drug carrier was also investigated. In a typical case, a contraceptive drug, norethindrone (NET), was bonded to PHPA, and the drug sustained released as long as 120 days an in vitro test. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2411–2417, 2000