Diastereoselective Enzymatic Aldol Additions: L‐Rhamnulose and L‐Fuculose 1‐Phosphate Aldolases from E. coli

Abstract

Two bacterial aldolases, RhuA and FucA, which are readily accessible by overex‐pression, catalyze the asymmetric addition of dihydroxyacetone phosphate to various aldehydes. The high degree of enantio‐and diastereoselectivity for the L‐threo (RhuA) and D‐erythro (FucA) stereochemistry of the products and the high stability of the enzymes make it possible to prepare rare carbohydrates and their derivatives as well as other polyhydroxylated compounds.

Quo vadis photorespiration: A tale of two aldolases

“…RhuA, FucA, and FruA from E. coli were purified as previously described [17]. Enzymes were freed of their endogenous metal (Zn 2+) by exhaustive dialysis [17].…”

“…Enzymes were freed of their endogenous metal (Zn 2+) by exhaustive dialysis [17]. After reconstitution of apo-RhuA or apoFucA with Co 2+, it was no longer possible to remove this metal from either enzyme by dialysis.…”

“…Each assay (1 ml) contained various concentrations of the substrates for these enzymes (or 1 mM L-rhamnulose 1-phosphate, L-fuculose 1-phosphate, or D-fructose 1,6-bisphosphate, respectively, for routine assay), 0.2 mM NADH, and a mixture of triosephosphate isomerase and glycerol phosphate dehydrogenase (10 units, Sigma Chemical Co.) [17]. Production of dihydroxyacetone phosphate in these coupled assays was monitored by loss of absorbance at 340 nm (e = 6220 cm -1 M -1) due to oxidation of NADH.…”

“…To 0.99 ml of 1 mM dihydroxyacetone phosphate, 50 mM MopsNaOH, pH 7, and 60 mM KC1, was added 10 I11 ofa 5 I.tM solution of RhuA that had been freed of metal by dialysis [17]. For assays that contained Co 2÷ as the activating metal, 10 I.tM Co 2+ was added to the final assay mixture.…”

“…Several metal-dependent (class II) aldolases exist that utilize a similar ene-diolate intermediate for comparable carboncarbon ligation chemistry. We have examined three such enzymes: L-rhamnulose 1-phosphate aldolase (RhuA), L-fuculose 1-phosphate aldolase (FucA), and D-fructose 1,6-bisphosphate aldolase (FruA) isolated from Escherichia coli [17], for their ability to catalyze oxygen-consuming side reactions.…”