Mechanistic studies on phosphoenol-pyruvate-utilizing enzymes involved in bacterial cell wall and lipopolysaccharide biosynthesis.

The study of the mechanisms of bacterial enzymes vital to the survival of the microorganisms were undertaken to lay the ground work for the development of mechanistically diverse and novel antimicrobial agents. The two enzymes used in this study, 3-deoxyoctulosonate 8-phosphate synthase (KDO 8-P synthase) and UDP-N-acetylglucosamine enolpyruvyl transferase (EPTase), both utilize phosphoenolpyruvate (PEP) as a substrate in catalysis. (Z)- and (E)- (3-$\sp2$H) PEP were converted into KDO 8-Ps deuterated at the C-3 position by incubation with unlabeled scD-arabinose 5-phosphate (A 5-P) in the presence of KDO 8-P synthase. Analysis of the stereochemistry of the two KDO 8-Ps deuterated at the C-3 position by $\sp1$H-NMR showed that the (Z)- (3-$\sp2$H) PEP had produced (3-$\sp2$H) KDO 8-P of predominantly the 3S configuration and the E isomer had given predominantly (3-R) (3-$\sp2$H) KDO 8-P. The results indicate a si face attack from the C-3 of PEP upon the re face of the carbonyl carbon of A 5-P. (2-$\sp{13}$C) PEP specifically labeled with $\sp{18}$O in the enolic oxygen position was incubated with KDO 8-P synthase in the presence of A 5-P to determine the fate of the enolic oxygen during catalysis. All of the $\sp{18}$O label was liberated as inorganic phosphate (Pi) as determined by heteronuclear shifted $\sp{31}$P and $\sp{13}$C NMR analysis of the reaction mixture. Similarly, KDO 8-P isolated from a reaction mixture of (2-$\sp{13}$C) PEP and A 5-P with KDO 8-P synthase in ($\sp{18}$O) H$\sb2$O was shown to contain $\sp{18}$O in the anomeric position. The results indicate that KDO 8-P synthase catalysis occurs with C-O bond cleavage of the PEP enolic bond with incorporation of oxygen from water into the anomeric position of KDO 8-P. EPTase catalysis was shown to occur with incorporation of deuterium from $\sp2$H$\sb2$O into the carboxyvinyl moiety of UDP-N-acetylglucosamine-3-enolpyruvate (UDP-GlcNAc-EP) in both the forward (UDP-GlcNAc + PEP) and reverse (UDP-GlcNAc-EP; Pi absent) directions. The enzyme also recognized both (E)- and (Z)-phosphoenolbutyrate (MePEP) as substrates, each analog yielding a mixture of geometric isomers of UDP-GlcNAc-3-enolbutyrate (UDP-GlcNAc-EB). 3-Methylphosphoenolbutyrate was not a substrate for EPTase. The geometric distribution of UDP-GlcNAc-EB was 75:25 (E:Z) from the (E)-MePEP reaction and 25:75 (E:Z) from the (Z)-MePEP reaction, indicating that catalysis occurs with some degree of stereospecificity. Enzymatic catalysis with either (E)- or (Z)-MePEP in $\sp2$H$\sb2$O yielded a 56:44 (E:Z) geometric distribution of nucleotide products. (2-$\sp{13}$C) and (3-$\sp{13}$C) PEP, when incubated with 1 mM EPTase, in the absence of UDP-GlcNAc, were converted into their respective labeled (R)-3-phosphoglycerates (3-PGA). Upon the addition of UDP-GlcNAc the $\sp{13}$C labeled 3-PGA was converted into UDP-GlcNAc-EP as determined by $\sp{13}$C NMR.PhDOrganic chemistryPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129262/2/9423178.pd