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Journal of Bacteriology, October 2008, p. 6686-6696, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00585-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process ^,

Yong Chang, Gary E. Wesenberg, Craig A. Bingman, and Brian G. Fox^*

Biophysics Graduate Degree Program and Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706-1549

Received 28 April 2008/ Accepted 11 August 2008

DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A ⁹ desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His₆ or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that 10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

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* Corresponding author. Mailing address: Department of Biochemistry, 433 Babcock Drive, Madison, WI 53706. Phone: (608) 262-9708. Fax: (608) 262-3453. E-mail: bgfox{at}biochem.wisc.edu

Published ahead of print on 22 August 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, October 2008, p. 6686-6696, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00585-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.