Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase
The enoyl reductase from <i>Mycobacterium tuberculosis</i> (<i>Mt</i>InhA) was shown to be a major target for isoniazid, the most prescribed first-line anti-tuberculosis agent. The <i>Mt</i>InhA (EC 1.3.1.9) protein catalyzes the hydride transfer from the 4<i&g...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-03-01
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| Series: | Future Pharmacology |
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| Online Access: | https://www.mdpi.com/2673-9879/3/2/23 |
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| author | Leonardo Kras Borges Martinelli Mariane Rotta Cristiano Valim Bizarro Pablo Machado Luiz Augusto Basso |
| author_facet | Leonardo Kras Borges Martinelli Mariane Rotta Cristiano Valim Bizarro Pablo Machado Luiz Augusto Basso |
| author_sort | Leonardo Kras Borges Martinelli |
| collection | DOAJ |
| description | The enoyl reductase from <i>Mycobacterium tuberculosis</i> (<i>Mt</i>InhA) was shown to be a major target for isoniazid, the most prescribed first-line anti-tuberculosis agent. The <i>Mt</i>InhA (EC 1.3.1.9) protein catalyzes the hydride transfer from the 4<i>S</i> hydrogen of β-NADH to carbon-3 of long-chain 2-<i>trans</i>-enoyl thioester substrates (enoyl-ACP or enoyl-CoA) to yield NAD<sup>+</sup> and acyl-ACP or acyl-CoA products. The latter are the long carbon chains of the meromycolate branch of mycolic acids, which are high-molecular-weight α-alkyl, β-hydroxy fatty acids of the mycobacterial cell wall. Here, stopped-flow measurements under single-turnover experimental conditions are presented for the study of the transient of reactants. Single-turnover experiments at various enzyme active sites were carried out. These studies suggested isomerization of the <i>Mt</i>InhA:NADH binary complex in pre-incubation and positive cooperativity that depends on the number of enzyme active sites occupied by the 2-<i>trans</i>-dodecenoyl-CoA (DD-CoA) substrate. Stopped-flow results for burst analysis indicate that product release does not contribute to the rate-limiting step of the <i>Mt</i>InhA-catalyzed chemical reaction. The bearings that the results presented herein have on function-based anti-tuberculosis drug design are discussed. |
| first_indexed | 2024-03-11T02:26:51Z |
| format | Article |
| id | doaj.art-e6d6dbf54ff241988628f0712d1dedee |
| institution | Directory Open Access Journal |
| issn | 2673-9879 |
| language | English |
| last_indexed | 2024-03-11T02:26:51Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Future Pharmacology |
| spelling | doaj.art-e6d6dbf54ff241988628f0712d1dedee2023-11-18T10:30:38ZengMDPI AGFuture Pharmacology2673-98792023-03-013237939110.3390/futurepharmacol3020023Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl ReductaseLeonardo Kras Borges Martinelli0Mariane Rotta1Cristiano Valim Bizarro2Pablo Machado3Luiz Augusto Basso4Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre 90619-900, BrazilCentro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre 90619-900, BrazilCentro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre 90619-900, BrazilCentro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre 90619-900, BrazilCentro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre 90619-900, BrazilThe enoyl reductase from <i>Mycobacterium tuberculosis</i> (<i>Mt</i>InhA) was shown to be a major target for isoniazid, the most prescribed first-line anti-tuberculosis agent. The <i>Mt</i>InhA (EC 1.3.1.9) protein catalyzes the hydride transfer from the 4<i>S</i> hydrogen of β-NADH to carbon-3 of long-chain 2-<i>trans</i>-enoyl thioester substrates (enoyl-ACP or enoyl-CoA) to yield NAD<sup>+</sup> and acyl-ACP or acyl-CoA products. The latter are the long carbon chains of the meromycolate branch of mycolic acids, which are high-molecular-weight α-alkyl, β-hydroxy fatty acids of the mycobacterial cell wall. Here, stopped-flow measurements under single-turnover experimental conditions are presented for the study of the transient of reactants. Single-turnover experiments at various enzyme active sites were carried out. These studies suggested isomerization of the <i>Mt</i>InhA:NADH binary complex in pre-incubation and positive cooperativity that depends on the number of enzyme active sites occupied by the 2-<i>trans</i>-dodecenoyl-CoA (DD-CoA) substrate. Stopped-flow results for burst analysis indicate that product release does not contribute to the rate-limiting step of the <i>Mt</i>InhA-catalyzed chemical reaction. The bearings that the results presented herein have on function-based anti-tuberculosis drug design are discussed.https://www.mdpi.com/2673-9879/3/2/23tuberculosisInhAenoyl reductasemycolic acidsisoniaziddrug target |
| spellingShingle | Leonardo Kras Borges Martinelli Mariane Rotta Cristiano Valim Bizarro Pablo Machado Luiz Augusto Basso Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase Future Pharmacology tuberculosis InhA enoyl reductase mycolic acids isoniazid drug target |
| title | Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase |
| title_full | Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase |
| title_fullStr | Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase |
| title_full_unstemmed | Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase |
| title_short | Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of <i>Mycobacterium tuberculosis</i> Enoyl Reductase |
| title_sort | single turnover of transient of reactants supports a complex interplay of conformational states in the mode of action of i mycobacterium tuberculosis i enoyl reductase |
| topic | tuberculosis InhA enoyl reductase mycolic acids isoniazid drug target |
| url | https://www.mdpi.com/2673-9879/3/2/23 |
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