FIG 3.

(A) Spectrophotometric changes during the transformation of maleate catalyzed by cell extracts of E. coli BL21(DE3) carrying pET28a-hbzIJ. Before the reaction started, 45 μg of cell extract was added to both the sample and the reference cuvettes containing 50 mM phosphate buffer (pH 7.4). The reaction was initiated by addition of 400 μM maleate. The spectra were recorded every minute after the addition of cell extracts. (B) Subsequent changes after the addition of 50 μg of purified d-malate dehydrogenase (DmlA), 1 mM DTT, 0.4 mM Mn²⁺, and 1 mM NAD⁺ into the sample and reference cuvettes after the reaction described for panel A. The formation of NADH (maximum λ, 340 nm) indicates the presence of d-malate in the product from HbzIJ-catalyzed maleate hydration.