Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase

Abstract

Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.

Crystal Structures of Staphylococcus aureus Ketol‐Acid Reductoisomerase in Complex with Two Transition State Analogues that Have Biocidal Activity

“…KARI is a bi‐functional enzyme catalyzing an isomerization reaction and an NAD(P)H‐mediated reduction. The enzyme has a strict requirement for Mg 2+ ions . Crystal structures of KARIs from different organisms have shown that both cofactors are located at the interface between the N‐ and C‐terminal domains of the enzyme .…”

Detection of Reaction Intermediates in Mg 2+ -Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography

“…This includes all DNA and RNA polymerases, helicases, nucleases, ligases, as well as ATP and GTP kinases and beyond. The method may be applied to studying enzymes that are not involved in nucleotide and nucleic acid metabolism, for example, (1) GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the Mg 2+ -dependent transfer of GalNAc from UDP-GalNAc to a serine or threonine residue in the first step of mucin O-linked glycosylation (Bennett et al , 2012), (2) Ketol-Acid Reductoisomerases (KARI), which require two Mg 2+ ions for the isomerization step in branched amino acid synthesis (Tadrowski et al , 2016), and (3) the radical SAM enzyme QueE, which utilizes Mg 2+ for radical-mediated ring contraction (Dowling et al , 2014). …”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…The metal ion affinities, determined by ITC, are similar to those of the well-characterised antibiotic-degrading MBL AIM-1 (72) (see also Chapter 5) and cyclic nucleotide diesterases, (257) but significantly weaker than those recorded for phosphotriesterases. (232) To date the in vivo metal ion composition of CpsB remains obscure, but Mn 2+ appears to be the most likely candidate, based on optimal catalytic performance combined with bioavailability.…”

“…The excess of Co 2+ was removed and fitted to a minimum number of Gaussian peaks to achieve the final composite spectrum using GRAMS AI software package. The data were analysed as described elsewhere (22,(218)(219)(220)(229)(230)(231)(232) .…”

“…The MCD spectrum of resting AIM-1 ( Figure 5.11) could be fit to no fewer than six transitions between 450 and 600 nm. Calculations using the angular overlap model (AOM) were used to assign the individual transitions as described in detail elsewhere (218,220,(229)(230)(231)(232) . The coordinates for the calculations were taken from the crystal structure (PDB code: 4AWY (216) ) and each Co 2+ site was treated separately.…”