Abstract
Genome sequencing showed that two proteins in Mycobacterium tuberculosis
H37Rv contain the metal binding motif (D/E)X(2)HX(approximately
100)(D/E)X(2)H 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 A 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 Delta9 desaturase from castor plant with an rms
difference 1.42 A. 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.
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