Figure 1.
Involvement of PKS12 Protein in the Biosynthesis of Mannosyl-β-1-Phosphomycoketide
A schematic representation of bimodular PKS12 protein from M. tuberculosis, which is required for the biosynthesis of mycoketide. The two modules of PKS12 are colored in blue (AT specificity for methylmalonyl CoA) and buff (AT specificity for malonyl CoA), and the N- and Cters are indicated in green and red, respectively. Individual domains are drawn as small boxes in the two modules. Mycoketide chain originates from repetitive condensations of methylmalonate and malonate units with medium-chain fatty acyl starters. This saturated chain is released from PKS12 by an unknown mechanism, which is followed by reduction, phosphorylation, and mannosylation to form MPM.
Figure 2.
Determination of Specificities of the AT Domains and Construction of Single-Module PKS12Δ1 Protein
(A) The PKS12Δ1 protein was constructed by deleting module 1 from the clone encoding PKS12 protein. The DNA encoding KS domains from module 1 and module 2 contains a conserved BsaBI restriction endonuclease site, which facilitated module 1 deletion. PKS12Δ1 retains N- and Cters of PKS12 protein. PKS12 AT2 null mutant (PKS12AT2°) and PKS12Δ1 AT null mutant (PKS12ΔAT°), both mutated at the catalytic Ser residue were constructed by using standard SDM protocols. The mutations in the domains are marked in red.
(B) Labeling of PKS12 protein with radioactive extender units. Lane 1 shows the Coomassie-stained, purified PKS12 protein (431 kDa). The protein could be labeled with both 14C-MCoA (lane 2) and 14C-MMCoA (lane 3). AT2 (S2672A) point mutant of PKS12 is labeled by 14C-MMCoA (lane 5) and not by 14C-MCoA (lane 4) depicting specificity of module 1 for MMCoA and module 2 for MCoA.
(C) Labeling of PKS12Δ1 protein with radioactive extender units. Lanes 1 and 2 show the Coomassie-stained, purified PKS12 (431 kDa) and PKS12Δ1 protein (234 kDa). PKS12Δ1 protein could be radiolabeled with 14C-MCoA (lane 3) but not with14C-MMCoA (lane 4). AT (S2672A) mutant protein of PKS12Δ1 does not incorporate any radioactive label (lanes 5 and 6).
Figure 3.
Biochemical Characterization of PKS12 and PKS12Δ1 Proteins
(A)Radio-TLC characterization of PKS12 products. Lane 1 to 8 show the propionyl, hexanoyl, octanoyl, decanoyl, lauroyl, palmitoyl, stearoyl, and 9-hydroxy decanoyl N-acetyl cysteamine thioester primed products. No product is formed in the absence of protein (lane 9).
(B) Time course of product formation catalyzed by PKS12 protein. The error bars correspond to three independent experiments, and the data points are an average of all three.
(C) Radio-TLC characterization of PKS12Δ1 products. Lane 1 to 8 show the propionyl, hexanoyl, octanoyl, decanoyl, lauroyl, palmitoyl, stearoyl, and 9-hydroxy decanoyl N-acetyl cysteamine thioester primed products. No product is formed in the absence of protein (lane 9).
(D) Time course of product formation catalyzed by PKS12Δ1 protein. The error bars correspond to three independent experiments, and the data points are an average of all three.
Figure 4.
Radio-HPLC and Mass Spectrometry Characterization of PKS12Δ1 Products
(A) Characterization of products on HPLC. The radioactive product synthesized by PKS12Δ1 was analyzed on reverse-phase HPLC, and the elution times were determined by a radioactive detector. The reaction was primed with a hexanoyl starter that undergoes two carbon extensions with MCoA. The product peaks superimpose with radioactive standards. The traces are stacked with an x-axis offset for clarity.
(B) Autoradiograph of the HPLC-resolved PKS12Δ1 products that were concentrated and analyzed by radio-TLC. The Rf of various bands is C8 (0.468), C10 (0.476), C12 (0.492), C14 (0.507), C16 (0.523), and C18 (0.539).
(C) ESI-MS analysis of the 29.46-min HPLC peak. Molecular ion peak of 199.17 confirms the eluent to be lauric acid. Isotopic distribution is in concordance with radioactive substrates utilized in the assay.
(D) Tandem MS of [M-H]−1199.17 peak. The fragmentation pattern was similar to standard lauric acid corresponding to decarboxylation (154.93) and loss of water (181.17) from the parent ion.
(E) Tandem MS of the [M*-H]−1 14C peak of lauric acid 201.17 peak. The characteristic peaks show mass difference of 2, corresponding to 14C radioisotope.
Figure 5.
PKS12 Models and Identification of Interacting Residues
(A) The end-to-end orientation of PKS12 is shown. Module 1 of PKS12 is shown in cyan and module two in copper brown, except the KS and ACP domains. The KS domains are shown in green and the ACP domains in yellow.
(B) The active site residues of KS and ACP domains are shown in ball and stick, and the distances are shown.
(C) Model for the docking domains of PKS12 generated by SCRWL and energy minimized using CVFF force field of Insight II. The interacting residues of the N- and the Cters are shown.
Figure 6.
Sedimentation Velocity Analyses
Analysis of PKS12 (A), PKS12Δ1 (B), PKS13 (C), and PKS2 (D) by sedimentation velocity in the XL1-analytical ultracentrifuge in 50 mM phosphate buffer (pH 7.2). The data were analyzed to yield the integral distribution of sedimentation coefficients.
Figure 7.
Quantitative Radio-HPLC Analyses of Linker-Change and Single Amino Acid Linker Mutants of PKS12Δ1 and Effect of Concentration on PKS12Δ1 Catalysis
(A) The radioactive products synthesized by PKS12Δ1 and PKS12Δ1 mutant proteins were analyzed on reverse-phase HPLC, and the elution times were determined by using a radioactive detector. All reactions were primed by hexanoyl starter that undergoes two carbon extension(s) with MCoA. Trace for E20S mutant (blue) shows exact retention time while traces of all other mutant proteins are stacked with an x-axis offset for clarity; linker-change protein (red), H7D + E20S mutant (green), wild-type PKS12Δ1 (pink), and H7D mutant (black). Chain lengths of standard fatty acids eluting at these retention times are marked on these stacked traces. Value of 0.01 V is considered as background, based on the control experiments.
(B) Comparison of concentration dependent changes in the product profiles catalyzed by PKS12Δ1. Gradation in color indicates increase of protein concentration for each set of peaks, which in turn depict the product formed. At higher protein concentration, more of longer chain length products are synthesized, since there is a shift in equilibrium towards higher order multimeric species.
Figure 8.
Effect of Cter Peptide on PKS12Δ1 Catalysis and AFM Analysis of PKS12Δ1
(A) Effect of Cter peptide, Cter-Nter concatenated peptide, and BSA on PKS12Δ1 catalysis. Specific inhibition of enzymatic activity by C-ter peptide can be seen from lanes 1 to 5. C-terminus peptide competes with other PKS12Δ1 molecules and thus specifically inhibits product formation. BSA and the concatenated C- and N-ter linker, which in itself forms a stable docking complex, does not interfere with PKS12Δ1 catalysis.
(B) Radio-HPLC analysis of PKS12Δ1 reaction inhibited by five times molar excess of Cter peptide. The solid curve indicates the control run without any Cter peptide, and the dotted curve shows the profile for inhibited assay.
(C) AFM analysis of PKS12Δ1 showing multimeric organization.
Figure 9.
Sfp from B. subtilis was used to label ACP2Cter of PKS12 (lane 1 and 2) and a noncognate ACP protein, Rv1344, from M. tuberculosis with 14C hexanoyl CoA. Although ACP2Cter was able to transfer the label to PKS12Δ1 (lane 1), no transfer was seen when either the KS mutant of PKS12Δ1 was used (lane 2) or when a noncognate ACP with PKS12Δ1 was used (lane 3). Minus ACP control experiment did not show any label on PKS12 (lane 4); there was also no direct labeling of PKS12Δ1 observed (lane 5)
Figure 10.
Schematic Representation of Modularly Iterative Catalysis
Supramolecular organization of PKS12 protein formed by interactions between N- and Cters.