Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Abstract

DesA3 is a membrane-bound stearoyl-CoA Δ9 desaturase that produces oleic acid, a precursor of mycobacterial membrane phospholipids and triglycerides. DesA3 has sequence homology with other membrane desaturases including the presence of the eight His motif proposed to bind the diiron center active site. This family of desaturases function as multi-component complexes, and thus require electron transfer proteins for efficient catalytic turnover. Here we present evidence that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. For these studies, Rv3230c was expressed as a partially soluble protein in Escherichia coli; recombinant DesA3 was expressed in Mycobacterium smegmatis as a catalytically active membrane protein. The addition of E. coli lysates containing Rv3230c to lysates of M. smegmatis expressing DesA3 gave strong conversion of [1-14 C]-18:0-CoA to [1-14 C]-cis-Δ 9 -18:1-CoA and of [1-14 C]-16:0-CoA to [1-14 C]-cis-Δ 9 -16:1-CoA. Both M. tuberculosis proteins were required to reconstitute activity, as various combinations of control lysates lacking either Rv3230c or DesA3 gave minimal or no activity. Furthermore, the specificity of interaction between Rv3230c and DesA3 was implied by the inability of other related redox systems to substitute for Rv3230c. The reconstituted activity was dependent upon the presence of NADPH, could be saturated by increasing the amount of Rv3230c added and was also sensitive to the salt concentration in the buffer. The results are consistent with the formation of a protein-protein complex, possibly with electrostatic character. This work defines a multi-protein, acyl-CoA desaturase complex from M. tuberculosis H37Rv to minimally consist of a soluble Rv3230c reductase and integral membrane DesA3 desaturase. Further implications of this finding relative to the properties of other multi-protein iron enzyme complexes are discussed.Mycobacterium tuberculosis is a human pathogen that causes one of the world's deadliest diseases, tuberculosis (TB 1 ). Current estimates are that up to one-third of the world's population may be infected with M. tuberculosis and that over two million people die from TB-related diseases each year (1-4). TB has resurged, in part due to the appearance of multipledrug-resistant strains. Consequently, the development of new drugs and the reexamination of drugs formerly deemed effective have become major focuses of world-wide TB research (3)(4)(5). This intense effort has included genome sequencing (6), proteome functional analysis by micro-array approaches (7-11), the TB Protein Structure Initiative [(12), * To whom correspondence should be addressed: Department of Biochemistry, 433 Babcock Drive, Madison, WI 53706. Telephone: (608) . E-mail: bgfox@biochem.wisc.edu. † This work was supported by the National Institutes of Health Grant R01 GM-50853 to B.G.F.1 Abbreviations: AlkB, alkane ω-hydroxylase; DesA3, product of Mycobacterium tuberculosis H37Rv gene rv3229c, a...

Global Study of IS 6110 in a Successful Mycobacterium tuberculosis Strain: Clues for Deciphering Its Behavior and for Its Rapid Detection

“…Among them, at least three copies of IS6110 interrupt different described operons, i.e., Rv2349c (plcB and plcC), Rv2823c (Rv2824c and Rv2819c), and Rv3229c (Rv3230c and Rv3229c) (41)(42)(43), and the potential transcriptional influence on these operons is an interesting point to be studied.…”

“…Its product is a stearoyl-coenzyme A desaturase (DesA3) implicated in the synthesis of oleic acid and considered to be essential for intracellular growth in macrophages (44,45). In addition, DesA3 is a target of the secondline antituberculosis drug isoxyl, which was used together with isoniazid in multiple-drug therapy in the 1960s (43,44). Isoxyl inhibits the synthesis of oleic and mycolic acids (43) and was demonstrated to be effective against various clinical isolates of multidrug-resistant strains of M. tuberculosis (46).…”

“…In addition, DesA3 is a target of the secondline antituberculosis drug isoxyl, which was used together with isoniazid in multiple-drug therapy in the 1960s (43,44). Isoxyl inhibits the synthesis of oleic and mycolic acids (43) and was demonstrated to be effective against various clinical isolates of multidrug-resistant strains of M. tuberculosis (46). The finding of the insertion of IS6110 in the desA3 (Rv3229c) gene suggests that isoxyl would not be effective against the Zaragoza strain.…”

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

“…The X-ray structure of phthalate dioxygenase reductase is the representative structure for this clade and domain arrangement (PDB ID 2PIA, [25]). In one example reported so far, catalytic cross-reactivity was not observed across the clades, i.e., T4moF did not substitute for Rv3230c [26]. The crossreactivity within the clades has not been explicitly tested.…”

“…The X-ray structure of BZD is the representative structure for this clade and domain arrangement (PDB ID 1KRH, [24]). The other clade, with representatives phthalate dioxygenase reductase [25] and Rv3230c [26], has N-terminal flavinand NAD-binding domains and a C-terminal [2Fe-2S] domain. The X-ray structure of phthalate dioxygenase reductase is the representative structure for this clade and domain arrangement (PDB ID 2PIA, [25]).…”