Cloning and Characterization of CYP51 from <i>Mycobacterium avium</i>

Pietila, Michael P.; Vohra, Pawan K.; Sanyal, Bharati; Wengenack, Nancy L.; Raghavakaimal, Sreekumar; Thomas, Charles F.

doi:10.1165/rcmb.2005-0398oc

Cited by 13 publications

(7 citation statements)

References 43 publications

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“…At the dawn of the new millennium, the orthologous nature of the first prokaryotic CYP51-like gene in Mtb was confirmed phylogenetically [61] and biochemically [62, 63]. CYP51 is not unique to Mtb since it is conserved in the mycobacterium genus [64, 65] and other actinomycetes-related species (Table S1, supporting material). An ortholog was also found and characterized in an obligate methanotrophic gram-negative bacterium Methylococcus capsulatus [66].…”

Section: Structural and Biochemical Analysis Of Individual Enzymesmentioning

confidence: 99%

The Mycobacterium tuberculosis cytochrome P450 system

Ouellet

Johnston

Montellano

2010

Archives of Biochemistry and Biophysics

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Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M. tuberculosis genome revealed the existence of twenty cytochrome P450 enzymes, some of which are potential candidates for drug targeting. The recent burst of studies reporting microarray-based gene essentiality and transcriptome analyses under in vitro, ex vivo and in vivo conditions highlight the importance of selected P450 isoforms for M. tuberculosis viability and pathogenicity. Current knowledge of the structural and biochemical properties of the M. tuberculosis P450 enzymes and their putative redox partners is reviewed, with an emphasis on findings related to their physiological function(s) as well as their potential as drug targets. KeywordsMycobacterium tuberculosis; cytochrome P450; azole drugs; CYP51; CYP121; CYP130; CYP128; FprA; cyclodipeptide; sulfolipid Tuberculosis prevalence and importanceTuberculosis (TB) 1 has been a scourge of mankind for nearly as long as recorded history exists. Egyptian mummies show evidence of skeletal decay caused by TB and have been shown to harbor Mycobacterium tuberculosis (Mtb) DNA [1]. Hippocrates, in "Of the epidemics" written in 400 BC, describes phthisis, a disease that is consistent with TB. Phthisis is the Greek work for consumption, the term by which tuberculosis was known for many centuries. In the Seventeenth Century, Paul Bunyan labeled tuberculosis the "Captain of all these Men of Death", reflecting its preeminence as a cause of infectious death [2]. TB is still with us today as a major worldwide threat.The discovery of Mtb, the causative agent of TB, was announced by Robert Koch in a famous lecture on March 24, 1882 [3]. In his lecture Koch, who received the Nobel Prize in 1905 for his discoveries, reminded his audience that one in seven human beings died of tuberculosis. The fifty years following Koch's groundbreaking work led to the development of the antituberculosis drugs that we still use to treat this disease, starting with isoniazid. The availability of effective drugs, together with improvements in economics, medicine and public health, have led to a gradual decrease in the incidence of TB in industrialized nations. However, NIH Public Access Author ManuscriptArch Biochem Biophys. Author manuscript; available in PMC 2011 January 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript worldwide, TB continues to be a leading cause of death, as the World Health Organization fact sheet for 2008 estimates that there were 9.2 million new TB cases in 2006, 1.7 million people died of the disease in that year, one-third of the world's population carries the latent (dormant) form of the disease, and one in ten people infected with latent Mtb bacilli will become sick with active TB in their lifetime [4]. Furthermore, TB has again become a concern in...

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Section: Structural and Biochemical Analysis Of Individual Enzymesmentioning

confidence: 99%

The Mycobacterium tuberculosis cytochrome P450 system

Ouellet

Johnston

Montellano

2010

Archives of Biochemistry and Biophysics

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“…Multiple in vitro approaches have suggested that a transmembrane anchor is not always required for the catalytic activity of some microsomal preparations of cytochrome P450s (29,30). For example, M. tuberculosis CYP51 specifically converts detergent-solubilized lanosterol to its demethylated product (31). However, the in vivo role of the membrane anchor in catalysis by other CYP51s that use membrane-associated lanosterol has yet to be established.…”

Section: Significancementioning

confidence: 99%

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

283

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Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.M embrane proteins that span the lipid bilayer once constitute around 50% of all integral membrane proteins (1). Although monospanning membrane proteins carry out numerous key biological functions, including environmental sensing, organellespecific catalysis, and the regulation of cell morphology, only individual domains or subdomains are currently represented in the Protein Data Bank, and structural information about interactions between their transmembrane domains and extramembranous components is lacking. Cytochrome P450 proteins are prominent enzymes with orthologs found in all kingdoms of life. In eukaryotes, microsomal members of this major family of mixed-function mono-oxygenases contain a single transmembrane helix and can be grouped in two broad functional categories: biodefense, such as the first phase of xenobiotic detoxification, and core metabolism including reactions in sterol biosynthesis and fatty acid oxidation (2).The lanosterol 14α-demethylases or CYP51 enzymes, probably the most genetically ancient of the cytochrome P450 families, play a central role in cholesterol or ergosterol biosynthesis (3). CYP51s carry out three consecutive mono-oxygenase reaction cycles to remove the 14α-methyl group from lanosterol to yield 4,4-dimethyl-cholesta-8,14,24-trienol, a key precursor in cholesterol and ergosterol biosynthesis, releasing water and formic acid (3). Because of the key roles that CYP51s play in yeast, filamentous fungi, and some parasitic protozoa, these enzymes are therapeutic targets for antimicrobial agents, including fluconazole (FLC), voriconazole (VCZ), and itraconazole (ITC) (4). Fungal infections play an increasingly significant role in disease, impacting agriculture ecosystems and human health, especially in immunocompromised individuals (5-7) for whom antifungal resistance continually poses a threat (8). In humans CYP51 is being tested as a target for cholesterol-lowering drugs (9) and in antiangiogenic cancer therapies (10). A limited set of cytochrome P450 isoforms (1A2, 2C8, 2C...

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“…M. smegmatis CYP51 bound clotrimazole, econazole, and miconazole with a two-to threefold-higher affinity than CYP164A2 (Table 2), in contrast to itraconazole, ketoconazole, and voriconazole, for which CYP164A2 and CYP51 had similar affinities. CYP164A2-binding affinities for clotrimazole, (30).…”

Section: Figmentioning

confidence: 99%

Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae

Warrilow

Jackson

Parker

et al. 2009

Antimicrob Agents Chemother

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The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K d , 1.2 to 2.5 M); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.Leprosy has afflicted humanity through the ages and remains today a serious and disfiguring condition in communities throughout the developing world. Immunization (18) and multidrug treatment programs have been effective in reducing morbidity and mortality due to lepromatous leprosy, but the incidence of new infections, at 680,000 per annum (36), remains high. Genome sequencing projects have been completed for several mycobacterial species, including both Mycobacterium leprae, the etiologic agent of leprosy, and Mycobacterium tuberculosis. A common objective of these schemes is to identify potential targets for the development of novel antimycobacterial compounds. Unusually for bacteria, the actinomycetes such as mycobacteria and streptomycetes contain substantial numbers of genes encoding cytochromes P450 (CYPs) or P450 pseudogenes (6, 23). These enzymes carry out a wide range of monooxygenation reactions involved in biocatalysis, secondary metabolism, and detoxification. CYPs are ubiquitous throughout the eukaryotes but are relatively uncommon in prokaryotes, with most prokaryotes containing no CYP genes. The unexpected discovery in M. tuberculosis of an ortholog of CYP51 (3), the sterol 14␣-demethylase of eukaryotes (19), proved a major catalyst for investigations into the CYP complements (CYPomes) of mycobacteria, as CYP51 is the target for azole antifungal drugs that have been shown to possess antimycobacterial properties (1,5,15,35).Most azole antifungal compounds show selective targeting of CYP51, having an 8-to 128-fold-higher affinity for fungal CYP51s relative to the orthologous enzyme present in the host (29). These compounds act by inhibiting the fungal sterol biosynthesis pathway, which does not exist in mycobacteria. The abundance of P450 proteins in mycobacterial species...

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Cloning and Characterization of CYP51 from Mycobacterium avium

Cited by 13 publications

References 43 publications

The Mycobacterium tuberculosis cytochrome P450 system

The Mycobacterium tuberculosis cytochrome P450 system

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae

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