Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway

Abstract

Background:The bacterial ManNAc-6P 2-epimerase (NanE) is essential for sialic acid salvage. Results: Crystal structures of NanE in complex with substrate/product coupled to 1 H NMR kinetics on wild-type and variants elucidate the C2-epimerization mechanism. Conclusion: A single lysine catalyst acts as a one-base mechanism for the C2-epimerization reaction interconverting ManNAc-6P to GlcNAc-6P. Significance: A novel deprotonation/reprotonation mechanism involving a single flexible lysine is described.

Crystal structures and kinetic analyses ofN-acetylmannosamine-6-phosphate 2-epimerases fromFusobacterium nucleatumandVibrio cholerae

“…Gene-deletion studies of NanE in Vibrio cholerae and Staphylococcus aureus have shown growth-inhibitory effects in the presence of Neu5Ac (Olson et al, 2013). NanE is conserved in Gramnegative and Gram-positive bacteria and is not homologous in structure or mechanism to the mammalian UDP-N-acetylglucosamine 2-epimerases (North et al, 2014;Pé lissier et al, 2014;Campbell et al, 2000). This makes bacterial NanE an interesting target for potential antibacterial/antibiotic development.…”

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…The X-ray crystal structures of NanE from Clostridium perfringens with bound ManNAc-6-P (PDB 4UTU)a nd bound GluNAc-6-P (PDB 4UTW) have also been solved. [204] As was observed for the V. cholera structures, the substrate and the product epimers are bound as their open-chain forms with most of the interactions with the protein retained for both ligands. Superposition of the structureso fb ound ManNAc-6-P and GluNAc-6-P is shown in Figure3I.…”

“…(H) N-Acetylmannosamine6-phosphate (NMan6P,blue) and N-acetyl-d-glucosamine 6-phosphate (NGlu6P,green) as boundt oN anE from ClostridiumP erfringens (PDB 4UTU and 4UTW,respectively)a re shown. [204] (I) N-Acetylmannosamine6-phosphate(NMan6P,blue) and N-acetyl-d-glucosamine 6-phosphate (NGlu6P, green)a sb ound to NanE from Vibrio cholerae (PDB 5ZJN and 5ZJP,respectively) are shown. [205] (J) GDP-a-d-mannose (GDP-Man, blue) and GDP-b-l-galactose (GDP-Gal, green) as bound to the K217Aa nd Y174F variants of GME from A. thaliana,respectively,(PDB 2C5E and 2C5A,r espectively) are shown.…”

“…The enzyme exists as ah omodimer and belongs to the TIMbarrel( ( b/a) 8 )s uperfamily.I nterestingly,N anE is believed to utilize ao ne-base (Lys) mechanism to catalyze the deprotonation and protonation reactions via an enolatei ntermediate (Scheme15). [204,230] The X-ray crystal structures of NanE from Vibrio cholera with bound ManNAc-6-P( PDB 5ZJN) and bound GluNAc-6-P (PDB 5ZJP) were solved by modelling either ligand into the same data set to obtain final structures of the two complexes. [205] In both structures, the substrate and the product are bound as their open-chain forms, and most of the interactions with the protein are the same forb oth ligands.…”

“…Again, the e-NH 2 of Lys66 is located 2.5 from C2 and ideally situated to abstract the a-proton of ManNAc-6-P.H owever,t he antipodal orientation of the C2 a-proton of GluNAc-6-P directs the C a ÀHb onda way from the e-NH 2 group of Lys66 toward the g-COO À of Glu180. From as tructural standpoint, Glu180 could be ac andidate for the "second Brønsted base" to deprotonate GluNAc-6-P at C2.H owever,P Ølissier et al [204] found that the E180A variant exhibited ac atalytic efficiency (k cat /K M )t hat was reduced only 60-foldr elative to that of the wild-typee nzyme with ManNAc-6Pa st he substrate. Althought he authors suggested that there may be rotation about the C2ÀC3 bond so that the aldehydea nd N-acetyl groups could move to allow deprotonation of GluNAc-6-P by Lys66, such motion seems improbable considering that the positions of these groups differ little between the substrate and product complexes ( Figure 3I).…”