Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Role of Serine140 in the mode of action of Mycobacterium tuberculosis ß-ketoacyl-ACP Reductase (MabA).

BACKGROUND: Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis ß-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli. FINDINGS: Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data. CONCLUSIONS: The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.

Mycobacterium tuberculosis beta-ketoacyl-ACP reductase: alpha-secondary kinetic isotope effects and kinetic and equilibrium mechanisms of substrate binding.

Beta-ketoacyl-ACP reductase catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein to generate beta-hydroxyacyl-acyl carrier protein and NADP+, the second step of the fatty acid elongation system type II of bacteria, plants, and apicomplexan organisms. Here, a modified and more efficient purification protocol is reported for recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP reductase (MabA). The increase in alpha-secondary deuterium kinetic isotope effect values measured at pH 10 as compared to those obtained at pH 7 points to isotope- and pH-sensitive steps occurring concomitantly. Equilibrium and kinetic fluorescence studies demonstrate positive cooperativity in binding of NADPH to MabA, with two forms of free enzyme in solution. Equilibrium dialysis shows no cooperativity in acetoacetyl-CoA binding to the enzyme. Moreover, modest affinity loss occurs when the substrates bind to the monomer as compared to the dimer of MabA. A mechanism of substrate binding to MabA is proposed on the basis of the experimental data.

Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) reductase: kinetic and chemical mechanisms.

Beta-ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of beta-ketoacyl-ACP to generate beta-hydroxyacyl-ACP and NADP(+). In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C(4)-proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse (D)2(O)V/K and an increase in (D)2(O)V when [4S-(2)H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data.

Selection of an Escherichia coli host that expresses mutant forms of Mycobacterium tuberculosis 2-trans enoyl-ACP(CoA) reductase and 3-ketoacyl-ACP(CoA) reductase enzymes.

Tuberculosis (TB) still remains a worldwide health concern. Efforts to understand the complex biology of Mycobacterium tuberculosis, the causative agent of TB, are important for new antitubercular drug development. Despite the completion of the genome sequence and the development of new genetic tools to manipulate this organism, the availability of sufficient amounts of mycobacterial proteins still remains an essential and laborious step to study the biochemical features of this pathogen. The T7-RNA polymerase-based pET system has been largely employed to express mycobacterial proteins in Escherichia coli, but it presents some limitations. To overcome problems with unstable expression of an M. tuberculosis inhA-encoded enoyl reductase mutant protein and lack of expression of two mabA-encoded ketoacyl reductase mutants, a sub-population of E. coli BL21(DE3) host cells was selected from a small-opaque colony. This empirically selected host, named BL21(DE3)NH, allowed stable expression of these mutant proteins. Although the mechanism that led the BL21(DE3)NH host to express the recombinant mutant proteins remains unknown, the persistent phenotype points to a stable genetic switch. This genetic alteration resulted in a tight control of the highly processive T7 RNA polymerase. Moreover, the absolute requirement for IPTG to obtain protein expression in the BL21(DE3)NH host cells suggests that no inherent defect in the transcriptional activity of the T7 promoter is present. Empirical host selection requires no further genetic manipulation of recombinant plasmids and may represent a means of obtaining tailor-made E. coli strains that overcome toxic effects associated with heterologous protein expression.