Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria

Valérie Guillet,Ségolène Galandrin,Laurent Maveyraud,Simon Ladevèze,Vincent Mariaule,Cécile Bon,Nathalie Eynard,Mamadou Daffé,Hedia Marrakchi,Lionel Mourey

doi:10.1074/jbc.m115.712612

Abstract

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes fromMycobacterium tuberculosisandMycobacterium marinum Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.

Highlights

Plexity of the mycobacterial cell envelope and underlies key features of mycobacterial physiology
The corresponding FadD proteins in M. tuberculosis are of two types, 12 fatty acyl-AMP ligases (FAALs) and 22 fatty acyl-coenzyme A (CoA) ligases (FACLs) [11]
Biochemical and Enzymatic Characterization of Mycobacterial FadD32 Enzymes—We previously described the biochemical characterization of FadD32 from M. tuberculosis (MtFadD32) [13], and the use of the orthologous enzymes from M. smegmatis (MsFadD32, 74% sequence identity) and C. glutamicum (CgFadD32, 39% sequence identity) for comparative studies and the development of a high throughput screening assay for FadD32 activity [17]

Summary

Experimental Procedures

Plasmids—The cloning of the fadD32 genes from M. tuberculosis, M. smegmatis, and Corynebacterium glutamicum has been described elsewhere [13, 17]. For specific activity experiments (Table 1), the enzyme was first subjected to serial dilution in HEPES pH 7.5 (with protein concentrations of 12.5 to 800 nM for MtFadD32, MmFadD32, and CgFadD32 and from 2.5 to 160 nM for MsFadD32) and added to the reaction mixture containing 20 ␮M lauric acid (C12) as substrate and 2 mM ATP. Crystals of MsFadD32 with either AMPC12 or AMPC20 were grown by mixing equal volumes of protein/inhibitor solutions and reservoir solution composed of PEG 1000, in 100 mM Tris-HCl, pH 8.2 to 8.7 These crystals were ϳ100 ϫ 100 ϫ 30 ␮m in size and displayed diffraction to a maximum resolution of 3.3 Å with synchrotron beams. The atomic coordinates and structure factors (codes 5EY8 and 5EY9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ

Results

Discussion

Protein name