Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

Denise A Rozwarski,Catherine Vilchèze,Michele Sugantino,Robert Bittman,James C Sacchettini

doi:10.1074/jbc.274.22.15582

Abstract

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.

Highlights

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein
Mycolic acids are long chain ␣-alkyl-␤-hydroxy fatty acids, which are a major component of mycobacterial cell walls [10, 11]
Several reasons for this are as follows: (i) the vast majority of the mutations found in isoniazid-resistant clinical isolates are associated with the isoniazid activator (38 – 41); (ii) only one enoyl-ACP reductase is found in M. tuberculosis, unlike some of the other enzymes of bacterial FAS-II systems [42]; and (iii) the longer substrate chain length specificity of InhA distinguishes it from the enoylACP reductases from other sources, such as the enoyl-ACP reductase component of the human FAS-I system

Summary

C16 Fatty Acyl Substrate*

The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. Even though mutations within the inhA gene are known to facilitate isoniazid resistance (21, 38 – 41), InhA remains a good candidate for drug design Several reasons for this are as follows: (i) the vast majority of the mutations found in isoniazid-resistant clinical isolates are associated with the isoniazid activator (the KatG catalase-peroxidase) (38 – 41); (ii) only one enoyl-ACP reductase is found in M. tuberculosis, unlike some of the other enzymes of bacterial FAS-II systems [42]; and (iii) the longer substrate chain length specificity of InhA distinguishes it from the enoylACP reductases from other sources, such as the enoyl-ACP reductase component of the human FAS-I system. This compound is not a natural biological substrate of InhA; it and long chain fatty acyl derivatives of coenzyme A are functional substrates in vitro

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Sacchettini