Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Reiße, Steven; Garbe, Daniel; Brück, Thomas

doi:10.1016/j.biochi.2014.10.024

Cited by 9 publications

(12 citation statements)

References 31 publications

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“… 8.0 n.a. — NADH 6 Mr-KARI Meiothermus ruber LRPGSRN 240 20 1.09 0.98 4.6 54.5 0.084 NADPH 27 So-KARI Spinacea oleracea (spinach) LRKGSNS 101 1.7 0.95 1.33 9.4 782.4 0.012 NADPH 30 Se-KARI Slackia exigua LREGSSS 45 1.0 0.41 0.8 9 800 0.011 NADPH 20 Ec-KARI Escherichia coli LRKEAIAEKRAS 1075 41 0.3 3.6 0.3 88 0.003 NADPH 17 Ec-KARI 6E6 Escherichia coli LRKE S IAEK D A D 30 650 2.3 0.20 74 0.40 185 NADH 17 Se-KARI DD Slackia exigua LREG D S D 113 880 0.97 0.10 9 0.11 81.8 NADH 20 Se-KARI DDV Slackia exigua LREG D S D 47 >1000 1.01 ...…”

Section: Resultsmentioning

confidence: 99%

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

Chen

Lin

et al. 2018

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 99%

“…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. Prior to the characterization of several native NADH-dependent KARIs 7 , 8 , 27 , this family of enzymes was believed to be exclusively NADPH-dependent.…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Lin

et al. 2018

Sci Rep

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“…KARI catalyses the second step of the BCAA biosynthesis pathway. With k cat values for KARIs from different sources reported in the range of about 1 s −1 ,,, this step is the slowest in the pathway. The KARI‐catalysed reaction is also of interest in biofuel production as it can contribute to the conversion of pyruvate to isobutanol .…”

Section: Discussionmentioning

confidence: 98%

“…Ac ombination of steady-state kinetics, [4,9,11] isotope effects, [15] site-directed mutagenesis [4] and crystallographic studies, [5a-c,e, 6] mainly on E. coli ands pinachK ARI, has led to the proposal that both the isomerisation and reduction reactions requiret wo metal ions and NADPH, and these need to be present in the same active site, since the isomerisation reaction alone is thermodynamically unfavourable (i.e.,t he equilibrium of the reaction is strongly on the side of the substrate). Indeed,n oi ntermediate of the reactioni sb eing released, despite the observation that the product of the isomerization reaction (i.e.,3 -hydroxy-3-methyl/ethyl-2-ketobutyrate) is an excellent substrate, but with relatively modest binding affinity.…”

Section: Discussionmentioning

confidence: 99%

“…[5a, 10a] However,i np lant KARI the Ndomain in the apoenzyme appears to have asignificant level of rotational freedom,b ut upon Mg 2 + andN ADPH binding the Na nd Cd omains are locked into place thus forming the recognition site for substrate binding. [5a] Based on the similarityo ft heir catalytic parameters (i.e., k cat values and Michaelis constants K m for substrates), [4,8,9,11] it appears likely that the structure of the catalytically competent enzyme-substrate complexa nd consequently the reactionm echanism is conserved in all KARIs.In this study,t he reactionm echanismso fE. coli and rice KARI are investigated using ar ange of physico-chemical techniques.…”

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

Tadrowski

Pedroso

Sieber

et al. 2016

Chemistry A European J

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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.

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Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

et al. 2016

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Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO ); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.

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Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Cited by 9 publications

References 31 publications

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

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

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

Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

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