RESUMO
Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.
Assuntos
Mycobacterium tuberculosis , Sistema Enzimático do Citocromo P-450/metabolismo , Colesterol/química , Descoberta de Drogas , Antituberculosos/farmacologia , Antituberculosos/químicaRESUMO
CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach. Using the CoupledMoves protocol of Rosetta, a virtual library of mutants was designed to bind compactin in a pro-pravastatin orientation. By examining the frequency of occurrence of beneficial substitutions and rational inspection of their interactions, a small set of eight mutants was predicted to show the desired selectivity and these variants were tested experimentally. The best CYP105AS1 variant gave >99% stereoselective hydroxylation of compactin to pravastatin, with complete elimination of the unwanted 6-epi-pravastatin diastereomer. The enzyme-substrate complexes were also examined by ultrashort molecular dynamics simulations of 50 × 100 ps and 5 × 22 ns, which revealed that the frequency of occurrence of near-attack conformations agreed with the experimentally observed stereoselectivity. These results show that a combination of computational methods and rational inspection could improve CYP105AS1 stereoselectivity beyond what was obtained by directed evolution. Moreover, the work lays out a general in silico framework for specificity engineering of enzymes of known structure.
RESUMO
There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antituberculosos/farmacologia , Desenho de Fármacos , Humanos , Tuberculose/tratamento farmacológico , Raios XRESUMO
CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3's high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Inativação MetabólicaRESUMO
A series of analogues of cyclo(l-tyrosyl-l-tyrosine), the substrate of the Mycobacterium tuberculosis enzyme CYP121, have been synthesized and analyzed by UV-vis and electron paramagnetic resonance spectroscopy and by X-ray crystallography. The introduction of iodine substituents onto cyclo(l-tyrosyl-l-tyrosine) results in sub-µM binding affinity for the CYP121 enzyme and a complete shift to the high-spin state of the heme FeIII. The introduction of halogens that are able to interact with heme groups is thus a feasible approach to the development of next-generation, tight binding inhibitors of the CYP121 enzyme, in the search for novel antitubercular compounds.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptídeos/química , Dipeptídeos/metabolismo , Halogenação , Mycobacterium tuberculosis/enzimologia , Sistema Enzimático do Citocromo P-450/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The emergence of untreatable drug-resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (K D). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert-butyl (10 g) compounds displaying optimal activity (MIC 1.562â µg/mL, K D 0.22â µM (10 f) and 4.81â µM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H-bond acceptors/H-bond donors 4/0, number of rotatable bonds 5-6, molecular volume >340â Å3, topological polar surface area <40â Å2.
RESUMO
The rise in multidrug resistant (MDR) cases of tuberculosis (TB) has led to the need for the development of TB drugs with different mechanisms of action. The genome sequence of Mycobacterium tuberculosis (Mtb) revealed twenty different genes coding for cytochrome P450s. CYP121A1 catalyzes a CC crosslinking reaction of dicyclotyrosine (cYY) producing mycocyclosin and current research suggests that either mycocyclosin is essential or the overproduction of cYY is toxic to Mtb. A series of 1,4-dibenzyl-2-imidazol-1-yl-methylpiperazine derivatives were designed and synthesised as cYY mimics. The derivatives substituted in the 4-position of the phenyl rings with halides or alkyl group showed promising antimycobacterial activity (MIC 6.25⯵g/mL), with the more lipophilic branched alkyl derivatives displaying optimal binding affinity with CYP121A1 (iPr KDâ¯=â¯1.6⯵M; tBu KDâ¯=â¯1.2⯵M). Computational studies revealed two possible binding modes within the CYP121A1 active site both of which would effectively block cYY from binding.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptídeos/química , Dipeptídeos/farmacologia , Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Antituberculosos/síntese química , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Dipeptídeos/síntese química , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/síntese química , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Tuberculose/tratamento farmacológicoRESUMO
Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to bind and oxidize diverse compounds, including steroids, terpenes and various human drugs. The wild-type P450 BM3 enzyme binds inefficiently to many azole antifungal drugs. However, we show that the BM3 A82F/F87V double mutant (DM) variant binds substantially tighter to numerous azole drugs than does the wild-type BM3, and that their binding occurs with more extensive heme spectral shifts indicative of complete binding of several azoles to the BM3 DM heme iron. We report here the first crystal structures of P450 BM3 bound to azole antifungal drugs - with the BM3 DM heme domain bound to the imidazole drugs clotrimazole and tioconazole, and to the triazole drugs fluconazole and voriconazole. This is the first report of any protein structure bound to the azole drug tioconazole, as well as the first example of voriconazole heme iron ligation through a pyrimidine nitrogen from its 5-fluoropyrimidine ring.
Assuntos
Antifúngicos/química , Azóis/química , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise Espectral , Relação Estrutura-AtividadeRESUMO
The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and its position in the active site, the P450 can catalyze a wide range of reactions including, e.g., hydroxylation, reduction, decarboxylation, sulfoxidation, N- and O-demethylation, epoxidation, deamination, CC bond formation and breakage, nitration, and dehalogenation. In this chapter, we describe the structural, biochemical, and catalytic properties of the P450s, along with spectroscopic and analytical methods used to characterize P450 enzymes and their redox partners. Important uses of P450 enzymes are highlighted, including how various P450s have been exploited for applications in synthetic biology.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Engenharia de Proteínas/métodos , Animais , Bactérias/química , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/química , Fungos/enzimologia , Fungos/genética , Fungos/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Oxirredução , Conformação Proteica , Biologia Sintética/métodosRESUMO
The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) are bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member - CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152K6 was characterized using spectroscopic, analytical and structural methods. CYP152K6, like its peroxygenase counterpart P450SPα (CYP152B1) from Sphingomonas paucimobilis, does not undergo significant fatty acid-induced perturbation to the heme spectrum, with the exception of a minor Soret shift observed on binding dodecanoic acid. However, CYP152K6 purified from an E. coli expression system was crystallized and its structure was determined to 1.3â¯Å with tetradecanoic acid bound. No lipids were present in conditions used for crystallogenesis, and thus CYP152K6 must form a complex by incorporating the fatty acid from E. coli cells. Turnover studies with dodecanoic acid revealed several products, with 2-hydroxydodecanoic acid as the major product and much smaller quantities of 3-hydroxydodecanoic acid. Secondary turnover products were undec-1-en-1-ol, 2-hydroxydodec-2-enoic acid and 2,3-dihydroxydodecanoic acid. This is the first report of a 2,3-hydroxylated fatty acid product made by a peroxygenase P450, with the dihydroxylated product formed by CYP152K6-catalyzed 3-hydroxylation of 2-hydroxydodecanoic acid, but not by 2-hydroxylation of 3-hydroxydodecanoic acid.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Ácidos Graxos/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Hidroxilação , Especificidade por SubstratoRESUMO
The cytochromes P450 (P450s or CYPs) constitute a large heme enzyme superfamily, members of which catalyze the oxidative transformation of a wide range of organic substrates, and whose functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The P450 peroxygenases are a subgroup of the P450s that have evolved in microbes to catalyze the oxidative metabolism of fatty acids, using hydrogen peroxide as an oxidant rather than NAD(P)H-driven redox partner systems typical of the vast majority of other characterized P450 enzymes. Early members of the peroxygenase (CYP152) family were shown to catalyze hydroxylation at the α and ß carbons of medium-to-long-chain fatty acids. However, more recent studies on other CYP152 family P450s revealed the ability to oxidatively decarboxylate fatty acids, generating terminal alkenes with potential applications as drop-in biofuels. Other research has revealed their capacity to decarboxylate and to desaturate hydroxylated fatty acids to form novel products. Structural data have revealed a common active site motif for the binding of the substrate carboxylate group in the peroxygenases, and mechanistic and transient kinetic analyses have demonstrated the formation of reactive iron-oxo species (compounds I and II) that are ultimately responsible for hydroxylation and decarboxylation of fatty acids, respectively. This short review will focus on the biochemical properties of the P450 peroxygenases and on their biotechnological applications with respect to production of volatile alkenes as biofuels, as well as other fine chemicals.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Peroxidases/metabolismo , Sequência de Aminoácidos , Biocombustíveis , Ácidos Carboxílicos/metabolismo , Catálise , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química , Ácidos Graxos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Hidroxilação , Oxirredução , Peroxidases/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH2-imidazole or CH2-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MICâ¯=â¯12.5⯵g/mL, 17a 50⯵g/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2â¯Å. A model generated from a 1.5â¯Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.
Assuntos
Antituberculosos/farmacologia , Azóis/farmacologia , Dipeptídeos/farmacologia , Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Piperazinas/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Azóis/química , Sítios de Ligação/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptídeos/química , Relação Dose-Resposta a Droga , Ligantes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Peptídeos Cíclicos/química , Piperazina , Piperazinas/química , Relação Estrutura-AtividadeRESUMO
Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short -CH2- linker displayed promising antimycobacterial activity, with the imidazole-CH2- series (7) showing low MIC values (6.25-25 µg/mL), which was also influenced by lipophilicity. Extending the linker to -C(O)NH(CH2)2- resulted in a loss of antimycobacterial activity. The binding affinity of the compounds with CYP121A1 was determined by UV-visible optical titrations with KD values of 2.63, 35.6, and 290 µM, respectively, for the tightest binding compounds 7e, 8b, and 13d from their respective series. Both binding affinity assays and docking studies of the CYP121A1 inhibitors suggest type II indirect binding through interstitial water molecules, with key binding residues Thr77, Val78, Val82, Val83, Met86, Ser237, Gln385, and Arg386, comparable with the binding interactions observed with fluconazole and the natural substrate dicyclotyrosine.
Assuntos
Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Mycobacterium tuberculosis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450/síntese química , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pirazóis/química , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrofotometria UltravioletaRESUMO
Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.
Assuntos
Avidina/química , Sistema Enzimático do Citocromo P-450/química , Dimetil Sulfóxido/farmacologia , Mycobacterium tuberculosis/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Dimetil Sulfóxido/química , Conformação Proteica , Desdobramento de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Jeotgalicoccus sp. 8456 OleTJE (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H2 O2 ) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleTJE . AldO oxidizes polyols (including glycerol), generating H2 O2 as a coproduct and facilitating its use for efficient OleTJE -dependent fatty acid decarboxylation. AldO activity is regulatable by polyol substrate titration, enabling control over H2 O2 supply to minimize oxidative inactivation of OleTJE and prolong activity for increased alkene production. We also use these fusion systems to generate novel products from secondary turnover of 2-OH and 3-OH myristic acid primary products, expanding the catalytic repertoire of OleTJE .
Assuntos
Oxirredutases do Álcool/metabolismo , Alcenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Peróxido de Hidrogênio/metabolismo , Microbiologia Industrial , Proteínas Recombinantes de Fusão/metabolismo , Oxirredutases do Álcool/genética , Biocatálise , Biocombustíveis , Sistema Enzimático do Citocromo P-450/genética , Descarboxilação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Engenharia Metabólica , Ácido Mirístico/metabolismo , Oxirredução , Proteínas Recombinantes de Fusão/genética , Staphylococcaceae/enzimologia , Staphylococcaceae/genética , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genéticaRESUMO
Similarity between the ligand binding profiles of enzymes may aid functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach for characterization of the ligand binding profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival, and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, whereas functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity between the fragment binding profiles of the orphan enzyme CYP144A1 and CYP121A1, a characterized enzyme that is important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, possible biological function, and provide the opportunity to develop inhibitors. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.
Assuntos
Proteínas de Bactérias/química , Inibidores das Enzimas do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/química , Mycobacterium tuberculosis/química , Filogenia , Proteínas Recombinantes/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Biologia Computacional , Inibidores das Enzimas do Citocromo P-450/classificação , Inibidores das Enzimas do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ligantes , Modelos Moleculares , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/enzimologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de ProteínaRESUMO
The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleTJE, indicating the key role of His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.
Assuntos
Alcenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Peroxidases/metabolismo , Staphylococcaceae/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Ácidos Graxos/metabolismo , Hidroxilação , Modelos Moleculares , Mutação , Peroxidases/química , Peroxidases/genética , Alinhamento de Sequência , Staphylococcaceae/química , Staphylococcaceae/genética , Staphylococcaceae/metabolismo , Especificidade por SubstratoRESUMO
TB, caused by the human pathogen Mycobacterium tuberculosis (Mtb), causes more deaths than any other infectious disease. Iron is crucial for Mtb to infect the host and to sustain infection, with Mtb encoding large numbers of iron-binding proteins. Many of these are hemoproteins with key roles, including defense against oxidative stress, cellular signaling and regulation, host cholesterol metabolism, and respiratory processes. Various heme enzymes in Mtb are validated drug targets and/or products of genes essential for bacterial viability or survival in the host. Here, we review the structure, function, and druggability of key Mtb heme enzymes and strategies used for their inhibition.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Hemeproteínas/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Humanos , Mycobacterium tuberculosis/metabolismoRESUMO
The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.