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

Chin-Yu Chen,Bo-Lin Lin,Tzu-Ping Ko,Jia-Cherng Horng,Kuan-Fu Lin,Cheng-Hung Chiang,Chun-Hsiang Huang

doi:10.1038/s41598-018-25361-4

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 KM 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.

Highlights

Isoleucine, leucine and valine are essential in the diets of animals because animals lack the machinery for producing these branched-chain amino acids (BCAAs)
Recombinant Sac-Ketol-acid reductoisomerase (KARI) was overexpressed in Escherichia coli BL21 (DE3) and purified by heating at 65 °C for 30 minutes followed by nickel-NTA affinity chromatography and gel filtration chromatography
This contrasts with KARI from Pseudomonas aeruginosa (Pa-KARI)[18], which forms a dodecamer, and Escherichia coli KARI (Ec-KARI)[19], which forms a tetramer, but is similar to the spinach (So-KARI)[22], Slackia exigua (Se-KARI)[20] and Mycobacterium tuberculosis (Mt-KARI)[13] enzymes, which are dimers

Summary

Introduction

Isoleucine, leucine and valine are essential in the diets of animals because animals lack the machinery for producing these branched-chain amino acids (BCAAs). KARIs with preference for the NADH cofactor are desirable for industrial applications, including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Based on multiple sequence alignment, information from available holo-formed structures[7,13,19,20,22,23,24,25,26], and engineering studies of KARIs17,20,21, Brinkmann-Chen et al showed that the acidic residues at conserved NADPH phosphate-binding positions in the β2-αB loop can be used to identify putative NADH-preferring KARIs in nature. We present the crystal structure of a class-I KARI from the thermoacidophilic archaeon Sulfolobus acidocaldarius at 1.75-Å resolution This enzyme, which we refer to here as Sac-KARI, was predicted to prefer NADPH based on the structure of its cofactor binding site. In addition to analysing the thermostability and acid tolerance of Sac-KARI by circular dichroism (CD) spectroscopy, we further investigate the temperature dependence of Sac-KARI activity and confirm its cofactor preference

Methods

Results

Conclusion