Fig 1.
Scheme of the penultimate step in the CoA biosynthetic pathway catalyzed by phosphopantetheine adenylyltransferase (PPAT).
PPAT reversibly transfers an adenylyl group from ATP to 4’-phosphopantetheine, producing 3’-dephospho-coenzyme A (dPCoA) and pyrophosphate (PPi).
Fig 2.
Multiple sequence alignment and phylogenetic analysis of MpaPPAT.
(A) Multiple sequence alignment of MpaPPAT with its homologues: Psychrophilic homologues, including MetPPAT (Methylocella tundrae) and MstPPAT (Methyloferula stellata); Mesophilic homologues, including EcoPPAT (Escherichia coli), MtuPPAT (Mycobacterium tuberculosis), and BsuPPAT (Bacillus subtilis); and Thermophilic homologues, including TthPPAT (Thermus thermophilus) and TmaPPAT (Thermotoga maritima). Highly conserved residues are highlighted in black, and partially conserved residues are shown in gray. The unique surface loop insertion (SCRLS in MpaPPAT) found in psychrophilic homologues is boxed in red. Positively and negatively charged residues lining the central funnel are indicated by blue and red diamonds, respectively. (B) Phylogenetic analysis of MpaPPAT homologues. The tree was constructed using the unrooted Neighbor-Joining method based on the alignment of the selected protein sequences.
Fig 3.
Overall crystal structure and hexameric assembly of MpaPPAT.
(A) Ribbon diagram of the MpaPPAT monomer, shown in two orientations related by a 90° rotation. α-helices (H1-H6) are colored green and β-strands (S1-S5) are yellow. The unique surface loop insertion (SCRLS, residues 67-71), located between H2 and S3, is highlighted in red. N- and C-termini are labeled. (B) Top view of the ‘dimer-of-trimers’ hexameric assembly. One monomer is colored as in panel A, while adjacent monomers are shown in gray. (C) Side view of the hexamer, illustrating the position of the SCRLS loop at the solvent-exposed surface and strand S5 at the interface.
Fig 4.
Structural comparison of the SCRLS loop region in MpaPPAT and its homologues.
(A) Comparison of the surface loop structure located between α-helix H2 and β-strand S3. The psychrophilic MpaPPAT (top left, superimposed with EcoPPAT) features a distinct loop insertion (SCRLS) highlighted in red. This extended loop is notably absent in mesophilic homologues, including EcoPPAT (gray), MtuPPAT (blue), and BsuPPAT (gold), as well as in thermophilic homologues TthPPAT (cyan) and TmaPPAT (purple). The PDB code for each structure was indicated.
Fig 5.
Functional and structural effects of the SCRLS loop deletion.
(A) Schematic representation of the two-step coupled assay used to quantify PPAT catalytic activity. (B) Comparative analysis of temperature-dependent activity between wild-type MpaPPAT (green) and the MpaPPAT(Δ67–71) mutant (cyan). The wild-type enzyme sustains high catalytic activity across a broad low-temperature range (10–30 °C), whereas the loop-deletion mutant shows markedly reduced activity, especially at 10 °C and 20 °C. Activity was quantified by monitoring NADPH absorbance at 340 nm in a two-stage coupled assay. Data are presented as mean S.D. (n = 3). (C) Structural superposition illustrating the effect of the loop deletion. The wild-type MpaPPAT (green) features the distinct, solvent-exposed loop element. In the MpaPPAT(Δ67–71) mutant (cyan), this entire loop is deleted, resulting in a short, truncated turn, as expected.
Fig 6.
Comparison of structural and electrostatic basis in the MpaPPAT and MpaPPAT(Δ67–71) mutant.
(A) Superposition of the hexameric core structures of wild-type MpaPPAT (green) and the MpaPPAT(Δ67–71) mutant (cyan). The close-up view highlights the conformational changes at the trimer-trimer interface, particularly around α-helix H4. The deletion of the distal SCRLS loop induces a long-range effect that stabilizes the H4-associated loop in the mutant (involving residues D92 and D95), leading to a “clamped” and rigidified core. An ATP molecule (stick model) from the EcoPPAT structure (PDB code 1GN8) is superimposed to illustrate the proximity of the active site to the clamped interface. (B) Electrostatic surface potential comparison of the central pore (viewed along the 3-fold axis). The wild-type enzyme (left) features a wide, positively charged (blue) central channel, essential for the electrostatic steering of negatively charged substrates. Conversely, the mutant (right) displays a constricted, negatively charged (red) pore, creating an electrostatic barrier that repels substrates. Blue and red represent positive and negative potentials, respectively (±5 kT/e).