Abstract
Biosynthesis of UDP-N-acetylmuramic acid in bacteria is a committed step towards peptidoglycan production. In an NADPH- and FAD-dependent reaction, the UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) reduces UDP-N-acetylglucosamine-enolpyruvate to UDP-N-acetylmuramic acid. We determined the three-dimensional structures of the ternary complex of Pseudomonas aeruginosa MurB with FAD and NADP+ in two crystal forms to resolutions of 2.2 and 2.1 Å, respectively, to investigate the structural basis of the first half-reaction, hydride transfer from NADPH to FAD. The nicotinamide ring of NADP+ stacks against the si face of the isoalloxazine ring of FAD, suggesting an unusual mode of hydride transfer to flavin. Comparison with the structure of the Escherichia coli MurB complex with UDP-N-acetylglucosamine-enolpyruvate shows that both substrates share the binding site located between two lobes of the substrate-binding domain III, consistent with a ping pong mechanism with sequential substrate binding. The nicotinamide and the enolpyruvyl moieties are strikingly well-aligned upon superimposition, both positioned for hydride transfer to and from FAD. However, flexibility of the substrate channel allows the non-reactive parts of the two substrates to bind in different conformations. A potassium ion in the active site may assist in substrate orientation and binding. These structural models should help in structure-aided drug design against MurB, which is essential for cell wall biogenesis and hence bacterial survival.
Highlights
Peptidoglycan (PG) is a vital component of the cell wall in most prokaryotic organisms
To produce the glycan building block UDP-Nacetylmuramic acid (UNAM), an enolpyruvyl group is transferred from phosphoenolpyruvate to UNAG by the UNAG enolpyruvyl transferase, MurA [5]
Structure determination and quality of the models The crystal structure of P. aeruginosa MurB (PaMurB) in complex with NADP+ was determined in two different crystal forms by molecular replacement
Summary
Peptidoglycan (PG) is a vital component of the cell wall in most prokaryotic organisms. The high molecular weight polymer provides osmotic stability and defines the size and shape of bacterial cells [1]. The cell wall PG of bacteria comprises a glycan chain of alternating N-acetyl-glucosamine and N-acetyl-muramic acid units, which are linked via peptide crosslinks. In Gramnegative bacteria, the formation of the high molecular weight PG polymer takes place in the periplasmic space, whereas the disaccharide-pentapeptide building blocks of PG are produced in the bacterial cytoplasm, linked to polyprenyl lipid chains and attached to the cytoplasmic membrane [2]. To produce the glycan building block UDP-Nacetylmuramic acid (UNAM), an enolpyruvyl group is transferred from phosphoenolpyruvate to UNAG by the UNAG enolpyruvyl transferase, MurA [5]. The pentapeptide stem is added in consecutive steps to the lactoyl moiety of UNAM [3,4]
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