scholarly journals The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

2005 ◽  
Vol 187 (23) ◽  
pp. 7970-7976 ◽  
Author(s):  
Mohini S. Ghatge ◽  
Kevin A. Reynolds
Keyword(s):  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Biosynthetic Gene Cluster ◽  
Conjugative Plasmid ◽  
Cytochrome P450 Monooxygenases ◽  
Phosphate Ester ◽  
High Sequence Similarity ◽  
P450 Monooxygenase ◽  
Complementation Experiment

ABSTRACT Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS 2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS 2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and k cat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.

scholarly journals In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

2021 ◽  
Vol 22 (15) ◽  
pp. 7811
Author(s):  
Olufunmilayo Olukemi Akapo ◽  
Joanna M. Macnar ◽  
Justyna D. Kryś ◽  
Puleng Rosinah Syed ◽  
Khajamohiddin Syed ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Structural Analysis ◽  
In Silico ◽  
Web Application ◽  
Cytochrome P450 Monooxygenases ◽  
Agglomerative Clustering ◽  
Ligand Complex ◽  
P450 Monooxygenase ◽  
Study Results

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.

scholarly journals Cytochrome P450 Monooxygenase CYP139 Family Involved in the Synthesis of Secondary Metabolites in 824 Mycobacterial Species

2019 ◽  
Vol 20 (11) ◽  
pp. 2690 ◽  
Author(s):  
Puleng Rosinah Syed ◽  
Wanping Chen ◽  
David R. Nelson ◽  
Abidemi Paul Kappo ◽  
Jae-Hyuk Yu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Secondary Metabolites ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Monooxygenase ◽  
Mycobacterial Species ◽  
P450 Monooxygenase ◽  
Physiological Capacity ◽  
Unique Amino Acid

Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis, is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily “A”. CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts’ metabolism and immune system.

scholarly journals Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

2016 ◽  
Vol 171 (4) ◽  
pp. 2458-2467 ◽  
Author(s):  
Naoyuki Umemoto ◽  
Masaru Nakayasu ◽  
Kiyoshi Ohyama ◽  
Mari Yotsu-Yamashita ◽  
Masaharu Mizutani ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases

scholarly journals Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

2005 ◽  
Vol 71 (11) ◽  
pp. 6968-6976 ◽  
Author(s):  
Volker Jungmann ◽  
István Molnár ◽  
Philip E. Hammer ◽  
D. Steven Hill ◽  
Ross Zirkle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Recombinant Proteins ◽  
Oxidation Reaction ◽  
Cytochrome P450 Monooxygenases ◽  
Enzyme Kinetic ◽  
Bacterial Strains ◽  
P450 Monooxygenase ◽  
P450 Monooxygenases

ABSTRACT 4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.

scholarly journals Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes

Plants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 577
Author(s):  
Mariko Baba ◽  
Ken-ichi Yamada ◽  
Michiho Ito
Keyword(s):  
Cytochrome P450 ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Volatile Component ◽  
Cloning And Expression ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Components ◽  
Cytochrome P450 Enzyme ◽  
Reaction Products ◽  
High Sequence Similarity

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.

scholarly journals Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium

2011 ◽  
Vol 64 (3) ◽  
pp. 221-227 ◽  
Author(s):  
Jie Hu ◽  
Hiroto Okawa ◽  
Kentaro Yamamoto ◽  
Kazuhiko Oyama ◽  
Masaaki Mitomi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Monooxygenase ◽  
Biosynthetic Gene ◽  
P450 Monooxygenase

scholarly journals The Isolation of Pyrroloformamide Congeners and Characterization of Its Biosynthetic Gene Cluster from Streptomyces sp. CB02980 Revealed a Unified Mechanism for Dithiolopyrrolone Biosynthesis

2019 ◽  
Author(s):  
Wenqing Zhou ◽  
Haoyu Liang ◽  
Xiangjing Qin ◽  
Danfeng Cao ◽  
Xiangcheng Zhu ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Biosynthetic Gene ◽  
High Sequence Similarity ◽  
Bicyclic Structure ◽  
Microbial Natural Products ◽  
Drug Leads

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. In the current study, two new dithiolopyrrolones, pyrroloformamide C (<b>3</b>) and pyrroloformamide D (<b>4</b>), were isolated from <i>Streptomyces </i>sp. CB02980, together with the known pyrroloformamides <b>1 </b>and <b>2</b>. The biosynthetic gene cluster for pyrroloformamides was identified from <i>S</i>. sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a non-ribosomal peptide synthetase, abolished the production of <b>1</b>－<b>4</b>. Overexpression of <i>pyfN</i>, a type II thioesterase gene, increased the production of <b>1</b> and <b>2</b>. The structure elucidation and biosynthetic characterization of pyrroloformamides <b>1</b> - <b>4</b> may inspire future efforts to discover new dithiolopyrrolones, which are promising drug leads for the treatment of infectious diseases or cancer.

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