Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

Abstract

Ketol‐acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched‐chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH‐dependent reduction of a 2‐ketoacid. The 2‐ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2‐ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2‐ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

The Chemical Mechanism of D‐1‐Deoxyxylulose‐5‐phosphate Reductoisomerase from Escherichia coli

“…Recent mechanistic and crystallographic investigations of KARI have revealed that two Mg 2+ ions are required at the active site. [18] The mechanism of the rearrangement catalyzed by KARI is generally accepted as being a concerted a-ketol reaction as there is no possibility of a retro-aldol/aldol sequence. An Mg 2+ ion is required for this isomerization step.…”

Regulation of Coenzyme A Biosynthesis in the Hyperthermophilic Bacterium Thermotoga maritima

“…As there is only one KPR that has been characterized from a hyperthermophile (17), it is difficult to judge, from a biochemical viewpoint, whether the catalytic efficiency of Tm-KPR/KARI is sufficient to conclude that it can act as (27). As T. maritima lacks the classical KPR gene, the relatively higher ratio of KPR activity against KARI activity in Tm-KPR/KARI than that in the E. coli KARI may reflect the fact that the protein does function as a KPR.…”

Eine Semipinakol‐Umlagerung – katalysiert durch ein Enzymsystem mit difunktioneller FAD‐abhängiger Monooxygenase

“…Unseres Wissens wurden lediglich zwei Enzyme beschrieben, die Semipinakol-Umlagerungen (Acyloin-Umlagerungen) katalysieren: [12][13][14][15][16][17] Acetohydroxysäure-Isomeroreduktase und 1-Desoxy-d-xylulose-5-phosphat-Reduktoisomerase sind an der Valin/Isoleucin-Biosynthese [12][13][14] bzw. dem 2-C-Methyl-d-erythritol-4-phosphat(MEP)-Biosyntheseweg beteiligt (Schema 2 B bzw.…”

“…Tyagi et al vermuten hierzu, dass die Reduktion für die sofortige Umsetzung und damit Stabilisierung des nicht begünstigten Intermediats notwendig ist. [13] …”

“…Für den Primärmetabo-lismus wurden lediglich zwei Beispiele bei der Biosynthese von verzweigten Aminosäuren und von 1-Desoxy-d-xylulose-5-phosphat beschrieben. [12][13][14][15][16][17] Die biosynthetische Umsetzung von Aurachin C (1) zu B (2) wurde erstmals mithilfe von Fütterungsexperimenten durch Hçfle und Kunze untersucht, [7] die die Herkunft der Hydroxygruppe an C-3 in Aurachin B (2) molekularem Sauerstoff zuordnen konnten. Kürzlich zeigten Pistorius et al, [8] dass es zusätzlich zum Genlocus, der für die Biosynthese des Aurachingrundgerüstes kodiert, zwei weitere Genloci gibt, die ebenfalls Gene der Aurachinbiosynthese enthalten.…”