Crystallization and preliminary X-ray crystallographic studies of fatty acid-CoA racemase fromMycobacterium tuberculosisH37Rv

Abstract

Fatty acid-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty-acid derivatives as it catalyzes the conversion of several (2R)-branched-chain fatty acid-CoAs to their (2S)-stereoisomers. Fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv has been purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 4000 as precipitant. The crystals belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 109.56, c = 147.97 A. The asymmetric unit contains six monomers, corresponding to a VM value of 2.15 A3 Da(-1). A complete native data set has been collected at 2.7 A resolution using a synchrotron-radiation source.

Crystal structure of fatty acid‐CoA racemase from Mycobacterium tuberculosis H37Rv

“…Protein expression, purification, and crystallization have been described elsewhere. 14 The crystals of the apo-protein were obtained by hanging-drop vapor-diffusion method at 295 K in 2 days. The reservoir solution used 20% (w/v) polyethylene glycol (PEG) 4,000, 0.05 M Tris-HCl, pH 8.5, 0.2 M ammonium chloride, and 0.1 M calcium chloride.…”

“…The structure of FAR was solved by a molecular replacement method using the Crystallography & NMR System (CNS) package 16 uted over the sequence. 14 The CNS was run using the data with a resolution range of 50.0 -2.7 Å in the space group P3 2 . Reflection data chosen randomly from 5% of the observed data were used for cross-validation with the R free value.…”

α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis—Detailed Kinetic and Structural Characterization of the Active Site

“…Previously, the crystal structure of MCR in the wild-type form (1.8 Å) and ligand-bound complexes (2–2.5 Å) have been reported, which have provided the general features of the enzyme molecule and some important structural insights on the active site and ligand interactions [ 11 , 12 , 13 ]. The crystal structure of the apo-form of another fatty acyl-CoA-racemase from M. tuberculosis has also been reported [ 14 , 15 ]. Here, we report the crystal structure of MCR in a new crystal form (the enzyme was expressed and purified using a modified protocol in Escherichia coli ( E. coli ) [ 16 ]) followed by the detailed structural and kinetic characterization of three key active-site mutants (H126A, D156A and E241A) with the aid of high-resolution crystal structures and a modified colorimetric assay, respectively.…”