Epimerization via Carbon−Carbon Bond Cleavage. l-Ribulose-5-phosphate 4-Epimerase as a Masked Class II Aldolase

Abstract

Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

Mechanistic insights into 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway

“…Actually, in this binding mode C3 of MEP which originates from C4 of DXP should be labeled by 18 O because the C4 hydroxyl group of DXP is not fixed by chelation with the DXR-bound M 2+ ; thus 18 O exchange occurs simultaneously upon the formation of the intermediate 7 (although it has been postulated that 7 might be a transient intermediate!) because it has been demonstrated that the compound 7 exists primarily in a hydrated form in aqueous solution [30,31]. But if the C3-C4 binding mode is operative, the phenomenon could be interpreted: the C4 hydroxyl group of DXP was confined by chelation with the DXRbound M 2+ , resulting in tight restriction of the retroaldol intermediate 7, which could entirely stop the exchange of its carbonyl oxygen atoms (Fig.…”

Enzymatic Characterization of 5-Methylthioribulose-1-phosphate Dehydratase of the Methionine Salvage Pathway inBacillus subtilis

“…23) In addition, MtnB is a homotetramer, as are the FucA, RibE, and RhuA enzymes. 14,18,24) These similarities suggest that MtnB is a new member of the FucA/ RibE/RhuA family and that these enzymes are related to each other evolutionally. It has been reported that production of DK-MTP-1-P using MTRu-1-P dehydratase from Klebsiella pneumoniae has been carried out in reaction buffer containing the magnesium ion, 25) and we used this information in our analysis of B. subtilis MtnB.…”

“…FucA, RibE, and RhuA are members of the divalent metal iondependent class II aldolase family. [13][14][15][16][17][18] These proteins, including MtnB, possess three histidine residues binding a metal ion, a glutamate or aspartate residue subtracting a proton from their substrates, and residues binding the phosphate group from the substrate (Fig. 8).…”

“…Our results suggest that MtnB requires metal ions for catalysis, but it is unclear what kind of metal ions bound to the MtnB purified from E. coli cells. Considering that FucA/RibE/RhuA-related enzymes require the zinc ion for optimal catalysis under physiological conditions and that recombinant class II aldolase enzymes expressed in E. coli cells were almost all of the Zn 2þ form, 14,26) purified MtnB might also be of the Zn 2þ form. The reaction mechanism of MtnB (MTRu-1-P dehydratase) was not examined in detail in this study.…”

“…[13][14][15][16][17][18] There has been no report on the dehydration activity of enzymes belonging to this family. In this, the enzymatic relationship of MtnB to this family of enzymes is discussed.…”