Abstract
Ketol-acid reductoisomerase (KARI) orchestrates the biosynthesis of branched-chain amino acids, an elementary reaction in prototrophic organisms as well as a valuable process in biotechnology. Bacterial KARIs belonging to class I organise as dimers or dodecamers and were intensively studied to understand their remarkable specificity towards NADH or NADPH, but also to develop antibiotics. Here, we present the first structural study on a KARI natively isolated from a methanogenic archaea. The dodecameric structure of 0.44-MDa was obtained in two different conformations, an open and close state refined to a resolution of 2.2-Å and 2.1-Å, respectively. These structures illustrate the conformational movement required for substrate and coenzyme binding. While the close state presents the complete NADP bound in front of a partially occupied Mg2+-site, the Mg2+-free open state contains a tartrate at the nicotinamide location and a bound NADP with the adenine-nicotinamide protruding out of the active site. Structural comparisons show a very high conservation of the active site environment and detailed analyses point towards few specific residues required for the dodecamerisation. These residues are not conserved in other dodecameric KARIs that stabilise their trimeric interface differently, suggesting that dodecamerisation, the cellular role of which is still unknown, might have occurred several times in the evolution of KARIs.
Highlights
Published: 11 November 2021The ketol-acid reductoisomerase (KARI, EC 1.1.1.86) catalyses the reversible conversion of acetohydroxy acids into the corresponding hydroxyl valerates, a crucial step in the biosynthesis pathway of valine, leucine and isoleucine [1]
MtKARI was initially purified by serendipity as a contaminant of the glutamine synthetase (GlnA) from cells grown under chemolithotrophic conditions at 65 ◦ C (Figure 1A) [23]
The protein was identified by MALDI-TOF-MS. This initial MtGlnA/MtKARI mixture was used for crystallisation and crystals of both proteins were obtained illustrating once more the selective power of crystallisation
Summary
The ketol-acid reductoisomerase (KARI, EC 1.1.1.86) catalyses the reversible conversion of acetohydroxy acids into the corresponding hydroxyl valerates, a crucial step in the biosynthesis pathway of valine, leucine and isoleucine [1]. The enzyme is necessary for amino acid prototrophy. Because of its importance in bacteria, plants and fungi, but its absence in metazoans, the enzyme receives increasing attention for the design of antibiotics, herbicides and fungicides [2,3,4]. On the other hand, branched-chain amino acids are valuable compounds for the food, cosmetic and pharmaceutical industries, with a constantly increasing global market (e.g., L-valine: average annual increase rate >5% [5,6]). Microbial fermentation is the first source of branched-chain amino acids worldwide. The study of the enzymes involved in their biosynthetic pathway is a far-reaching process, which explains the numerous characterised KARIs among prokaryotes
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