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Bacillus anthracis inosine 5'-monophosphate dehydrogenase in action: the first bacterial series of structures of phosphate ion-, substrate-, and product-bound complexes - PubMed

  • ️Sun Jan 01 2012

Bacillus anthracis inosine 5'-monophosphate dehydrogenase in action: the first bacterial series of structures of phosphate ion-, substrate-, and product-bound complexes

Magdalena Makowska-Grzyska et al. Biochemistry. 2012.

Abstract

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, inosine 5'-monophosphate (IMP), and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1

(A) Mechanism of the IMPDH reaction. The covalent enzyme–thioimidate intermediate is shown as E-XMP*. B. anthracis numbering is used. (B) Inhibitors targeting human IMPDH.

Figure 2
Figure 2

Multiple-sequence alignment of IMPDHs from selected bacterial pathogens. The following IMPDH sequences were used in the alignment: B. anthracis strain Ames, S. pyogenes, Streptococcus pneumoniae, Cl. perf ringens, Ca. jejuni, Vibrio cholerae, Haemophilus inf luenzae, Francisella tularensis, and Bo. burgdorferi. Identical residues are highlighted in red, and similar residues are shown as red letters. Secondary structure elements derived from B. anthracis IMPDH are depicted as arrows (representing β-strands) and cylinders (representing α-helices and 310-helices) and are numbered consecutively. The positions for the active site cysteine, hydrolysis dyad, and residues from the C. parvum signature motif are marked as black rectangles and labeled.

Figure 3
Figure 3

Structure of chain A of the B. anthracis IMPDH complex with IMP (gray space-filling model) showing the TIM barrel and CBS domains (left). Structure of the tetramer in a view parallel to the 4-fold axis (right). The tetramer was generated by performing the appropriate symmetry operation on the more complete monomer (chain A). Each subunit is shown in a different color with a space-filling model of IMP in the active site of each subunit. The inset above the tetramer shows the specific regions of the protein participating in the formation of the tetramer.

Figure 4
Figure 4

Structural alignment of IMPDH active sites. (A) Overlap of the B. anthracis IMPDH structures of Pi (green), IMP (magenta), and XMP (yellow) complexes showing the positions of the IMP and XMP (magenta and yellow sticks, respectively). (B) Overlap of the B. anthracis Pi-bound (green) and IMP-bound (magenta) structures with the S. pyogenes enzyme-IMP complex (PDB entry 1ZFJ) (turquoise). IMP molecules (B. anthracis in magenta, S. pyogenes in turquoise) are depicted as sticks. S. pyogenes residues Met393 and Gly394 interacting with IMP are also depicted as sticks. (C) Overlap of the structures of the B. anthracis Pi-bound enzyme (green) with the T. foetus IMPDH complex with MZP (purple) (PDB entry 1PVN). The conserved Arg-Tyr dyad within helix η 3 of the flap is depicted as sticks. (D) Overlap of the structures of the B. anthracis Pi-bound enzyme (green) with the T. foetus IMPDH (purple) complex with IMP (omitted for the sake of clarity) and TAD (purple) (PDB entry 1LRT). A distal portion of the B. anthracis catalytic flap with the conserved Arg-Tyr dyad is clashing with the T. foetus β-methylene-thiazole portion of TAD, indicating that these two elements are occupying the same space within the active site.

Figure 5
Figure 5

Different degrees of flap disorder in bacterial IMPDHs. (A) B. anthracis monomer (molecule A) of the highly ordered Pi-bound (apo) structure. (B) Detail of the active site, from left to right, of the B. anthracis Pi-bound enzyme (entire flap ordered), the S. pyogenes complex with IMP (missing residues 402–415 that include helix η3), and the B. anthracis complex with XMP (missing residues 394–414, including helices α15 and η3) and with IMP (missing residues 381–421), respectively. The catalytic flap is colored magenta, and the catalytic loop is colored dark blue, with the catalytic Cys represented as sticks. Ligands (Pi, IMP, and XMP) are colored red, orange, and green sticks, respectively.

Figure 6
Figure 6

Steady state kinetics of B. anthracis IMPDH. (A) Secondary plot of kinetic data showing apparent Vmax values (from initial velocity vs NAD+ plots) plotted vs IMP concentration. The solid line represents the best fit to eq 1 for Michaelis–Menten kinetics. (B) Secondary plot of kinetic data showing apparent Vmax values (from initial velocity vs IMP plots) plotted vs NAD+ concentration. The solid line represents the best fit to eq 2 for uncompetitive substrate inhibition.

Figure 7
Figure 7

Biolayer interferometry analysis using Octet RED of binding of A110 and C91 to B. anthracis IMPDH. The analysis was performed in triplicate. For each triplicate, the responses were fit to a 1:1 protein-ligand interaction model. The obtained values were used to generate steady state plots.

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