Figure 1.
MurB-catalyzed FAD- and NADPH-dependent reduction of UNAGEP to UNAM.
In the first half reaction (upper panel) the 4-pro-S hydrogen and two electrons from the reduced nicotinamide C4 atom are transferred to the N5 atom of the isoalloxazine resulting in NADP+ and reduced FAD. In the second half reaction (lower panel) the hydride is transferred to the C3 atom of the UNAGEP enolpyruvyl group, reducing the enolpyruvyl to a lactoyl group and thus converting UNAGEP to UNAM.
Table 1.
Data collection and structure refinement statistics.
Figure 2.
Structure-based sequence alignment of PaMurB against three MurB enzymes with experimentally determined crystal structures.
Each of the three enzymes represents one type of MurB. Annotations of secondary structure are based on PaMurB and are colored red for FAD-binding domain I, blue for domain II and pink for substrate-binding domain III. Strictly conserved residues are shaded in red and conserved regions are boxed; the GXG motif important for FAD-binding is indicated and shaded in yellow. The sequences compared are Pseudomonas aeruginosa MurB (PA2977), Escherichia coli MurB (EC(I), type I, PDB code 2MBR), Staphylococcus aureus MurB (SA(IIa), type IIa, PDB code 1HSK), and Thermus caldophilus MurB (TC(IIb), type IIb, PDB code 2GQT). Residues involved in cofactor- and substrate-binding are indicated: ⧫, residues that interact with FAD in both PaMurB and EcMurB; ▴, equivalent residues that interact with UNAGEP in EcMurB; □, residues that interact with NADP+;⊗, residues that interact with both UNAGEP and NADP+; ✶, residues that coordinate the putative catalytic metal ion.
Figure 3.
Overall structure of the ternary complex of PaMurB with FAD and NADP+.
A. Stereo view of the crystal structure of PaMurB with bound FAD and NADP+. The enzyme is shown in cartoon representation and comprises FAD-binding domain I (red) and domain II (blue), and the substrate-binding domain III (pink). FAD and NADP+ are shown as yellow and cyan stick models, respectively. NADP+ occupies the channel between the two lobes of domain III (in this view: left, lobe 1; right, lobe 2). Relevant secondary structure elements are labeled. B. Stereo view of the superimposition of the Cα traces of EcMurB (olive green), SaMurB (orange) and TcMurB (red) against PaMurB (black). PaMurB and EcMurB display highly similar structures in their respective NADP- and UNAGEP-bound complexes. In domain III, type II MurB enzymes lack the tyrosine loop preceding helices α4 and α5, as well as the protruding βαββ fold on lobe 2.
Figure 4.
NADP+ and the substrate binding site.
A. Stereo view of NADP+ bound in the substrate channel of PaMurB. NADP+ is depicted as a cyan stick model. The Fo-Fc omit electron density of NADP+, contoured at 3.0 σ, indicates tight binding of the ligand. The nicotinamide ring stacks against the isoalloxazine ring system of FAD (shown in yellow). Residues of the binding site that form hydrogen bonds with NADP+ include Tyr-132, Arg-166 and Glu-335 for the nicotinamide moiety, Lys-227 for the diphosphate backbone, and Tyr-196, Asn-243 and Lys-272 for the adenosine. The adenosine moiety is in addition stabilized by stacking interactions with Tyr-196 and Tyr-264. B and C. Comparison of protein residues interacting with NADP+ and UNAGEP (based on EcMurB, PDB code 2MBR). NADP+ and UNAGEP are shown as ball-and-stick models in cyan and green, respectively. Red radiating lines around ligand atoms indicate van der Waals contacts, while green dashes represent potential hydrogen bonds. Ball-and-stick models are shown for binding site residues that provide polar interactions but not those involved in van der Waals interactions only. Orange shading highlights binding site residues that are conserved and involved in binding both substrates.
Figure 5.
NADP+ shares the same substrate binding site with UNAGEP.
A. Stereo view of PaMurB (black ribbon) and UNAGEP-bound EcMurB (olive green ribbon, 2MBR) superimposed based on their FAD atomic coordinates. FAD, NADP+ and UNAGEP are depicted as stick models in yellow, cyan and green, respectively. NADP and UNAGEP occupy the same substrate channel located between the lobes of domain III. B. Co-localization of NADP(H) and UNAGEP substrate moieties on the si face of the FAD isoalloxazine ring system. The reactive moieties of the two substrates align together after superimposing the structures of PaMurB-NADP+ and EcMurB-UNAGEP (PDB code 2MBR) based on the FAD atomic coordinates. However, the non-reactive parts of the ligands diverge in the binding site, which can be visualized as three loci. S: the substrate moiety that reacts with FAD. B: the backbone region consisting of sugar and diphosphate. N: the non-reactive nucleotide moiety, which shows the greatest deviation. The remodeling of the binding site according to individual substrates is discussed in the text.
Figure 6.
Potassium binding site in PaMurB and its proposed role in catalysis.
A. Structure of the potassium binding site in PaMurB crystal form A. The coordination sphere is formed by the carboxamide oxygen of NADP+ nicotinamide in addition to two side chain oxygens and two main chain oxygens from the protein. The potassium ion (gold sphere) and its Fo-Fc omit difference density contoured at 5.0 σ (green mesh) are shown. B. The active site potassium ion assists in substrate orientation and binding. Superimposition of the PaMurB crystal form A structure and the EcMurB-UNAGEP complex (PDB code 2MBR) based on FAD atomic coordinates shows that the C2-C3-C4 locus of NADP+ nicotinamide (in cyan) spatially overlap with the enolpyruvyl group of UNAGEP (in green). Both substrate moieties are bound to Glu-335 and the backbone amine of Ser-239. The nicotinamide C4n atom (cyan sphere), which transfers a hydride to the isoalloxazine N5 atom, coincides with the enolpyruvyl C3e (green sphere), which receives the hydride during the second half-reaction. The geometric relation of the C4n atom to the isoalloxazine is indicated. In synergy with the isoalloxazine ring, Glu-335 and Ser-239, the potassium ion positions NADPH and UNAGEP such that the substrate carbons are in the optimal position for hydride transfer.