Identification, cloning, purification, and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.
The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).
Bailey
Ann Marie
AM
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Mahapatra
Sebabrata
S
Brennan
Patrick J
PJ
Crick
Dean C
DC
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase