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X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Department of Biochemistry, University of Wisconsin, Madison, Madison, Wisconsin 53706, USA

(RECEIVED December 13, 2004; FINAL REVISION February 10, 2005; ACCEPTED February 11, 2005)

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X₂HX_~100(D/E)X₂H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 Å resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP 9 desaturase from castor plant with an rms difference 1.42 Å. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.

Keywords: desaturase; Mycobacterium tuberculosis; X-ray crystallography

Abbreviations: A cis -5–24:1 fatty acid is a C₂₄ molecule with one cis double bond at the C–5 position • a cis-³, cis-¹⁵-34:2 fatty acid is a C₃₄ molecule with two cis double bonds at the C–3 and C–15 positions • IPTG, isopropyl--D-thiogalactopyranoside • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • OD₆₀₀, optical density at 600 nm • MAD, multiple anomalous dispersion • rms, root mean square.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041288005.

Reprint requests to: Brian G. Fox, Department of Biochemistry, 433 Babcock Dr., University of Wisconsin, Madison, Madison, WI 53706, USA; e-mail: bgfox{at}biochem.wisc.edu; fax: (608) 262-3453.

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