NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius

Abstract

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K _M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

Figure 1

Biosynthetic pathways for BCAA. In the first reaction acetolactate synthase (ALS) condenses one pyruvate with another into 2-acetoacetate (for valine and leucine) or with 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate (for isoleucine). Both products are then isomerized and reduced via keto-acid reductoisomerase (KARI) to form 2,3-dihydroxyisovalerate and 2,3-dihydroxy-3-methylvalerate, respectively. In this unusual two-step reaction, each substrate undergoes Mg²⁺-dependent alkyl migration followed by NAD(P)H-dependent reduction of the 2-keto group. Dihydroxyacid dehydratase (DHAD) converts two products of the second step to 2-ketoisovalerate and 2-keto-3-methylvalerate, respectively. The final step in the parallel pathway is conducted catalysed by amino transferase, which yields the final products of valine from 2-ketoisovalerate and isoleucine from 2-keto-3-methylvalerate. In contrast, a series of more additional enzymes are necessary to sequentially divert 2-ketoisovalerate to the leucine production pathway.

Figure 2

CD spectra of Sac-KARI in phosphate buffer (pH 8.0) containing 2 mM Mg²⁺. (A) The far-UV CD spectra of Sac-KARI measured over the wavelength range 200 to 260 nm at temperatures from 25 to 95 °C. (B) The far-UV CD spectra show that the secondary structure of Sac-KARI is unaffected by changes in pH over the range of pH values from 3.0 to 8.0. (C) CD-monitored thermal unfolding curve of Sac-KARI at 208 nm. Fitting of the thermal melting curve indicates that the Tm of Sac-KARI is approximately 86 °C.

Figure 3

Crystal structure of Sac-KARI. (A) A dimer comprising the asymmetric unit is shown as a ribbon model with the monomers coloured green and cyan. The N and C termini are indicated, as are the Rossmann and knot domains and a few secondary structural elements of interest, which are coloured yellow. The bound sulphate and magnesium ions are shown as stick-and-sphere models. (B) The second sulphate interacts with the β2-αB loop. It can form hydrogen bonds with R49 and S53, as does the 2′-phosphate of NADPH. (C) Magnesium binding. The bound magnesium ion is shown as a purple sphere. The surrounding acidic side chains are shown as stick models. D191 is from the second monomer, which is coloured cyan rather than green.