Essential Cysteines in 3-Deoxy-d-manno-octulosonic Acid 8-Phosphate Synthase from Escherichia coli:  Analysis by Chemical Modification and Site-Directed Mutagenesis

Abstract

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their e...

Substrate Ambiguity of 3-Deoxy- d - manno -Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae Revisited

“…The apparent molecular mass was determined by size-exclusion chromatography and was found to be 88.2 kDa. This value is similar to those reported for the KDO8P synthases from E. coli, Aquifex aeolicus, and Salmonella enterica serovar Typhimurium (3,(12)(13)(14) Taylor and Woodard, submitted).…”

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase

“…5). The other significant consumer of PEP during optimal growth on glucose is aromatic amino acid biosynthesis (~ 2%), with fluxes to cell wall and outer membrane biosynthesis having a combined contribution of only ~ 0.5% 11,12 .…”

The Gene Expression and Enzyme Activity of Plant 3-Deoxy-D-Manno-2-Octulosonic Acid-8-Phosphate Synthase Are Preferentially Associated with Cell Division in a Cell Cycle-Dependent Manner

“…Similarly, substituting H198, H242, and C164 in the tomato Kdo-8-P synthase greatly impairs the tomato enzyme activity. This suggests that these residues share the same function as their bacterial counterparts: H198 may behave as a general base in the catalytic mech- anism, establishing salt bridges between its positive charge and a phosphate oxygen of PEP (Sheflyan et al, 1999;Howe et al, 2000;Asojo et al, 2001); H242 may be involved in the maintenance of the correct local environment at the active site (Sheflyan et al, 1999); and C164 may be required for the normal folding of the protein (Salleh et al, 1996).…”

“…The amino acid residues shown to be essential for E. coli Kdo-8-P synthase activity (Salleh et al, 1996;Sheflyan et al, 1999;Radaev et al, 2000) are conserved in the sequence of the plant enzymes as shown in Figure 1. In the tomato Kdo-8-P synthase, residue K136 may be involved in the binding of PEP, residues R61 and T62 may be involved in the binding of Ara-5-P, and residues C164, H198, and H242 may represent structurally important residues for maintenance of active sites.…”