Uncovering rare NADH-preferring ketol-acid reductoisomerases

Brinkmann‐Chen, Sabine; Cahn, John W.; Arnold, Frances H.

doi:10.1016/j.ymben.2014.08.003

Cited by 27 publications

(45 citation statements)

References 35 publications

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“…The cofactor specificity of Aa_KARI from Alicyclobacillus acidocaldarius was inverted by the introduction of three mutations in its specificity loop [21]. Ua_KARI from an uncultured organism and Ia_KARI from Ignisphaera aggregans were correctly predicted to utilize NADH, based on their loop sequences [2]. The fourth KARI, from Azotobacter vinelandii, crystallized serendipitously as an impurity of another protein purified from A. vinelandii lysate.…”

Section: Resultsmentioning

confidence: 99%

“…Until recently, KARI family enzymes were believed to be exclusively NADPH-dependent [2]. Because NADH utilization is advantageous in the industrial production of amino acids and biofuels [21, 23], engineering KARI cofactor specificity has been of considerable interest.…”

Section: Resultsmentioning

confidence: 99%

“…Cloning, expression and purification of Aa_KARI ( Alicyclobacillus acidocaldarius ), Ua_KARI (Uncultured archaeon), and Ia_KARI ( Ignisphaera aggregans ) were described previously [2, 21]. Av_KARI was isolated from Azotobacter vinelandii strain AvOP (ATCC BAA-1303).…”

Section: Methodsmentioning

confidence: 99%

“…The tubes were incubated in their slots for 1 h, and then quenched on ice. Residual activity was determined with the activity assay described previously [2]. …”

Section: Methodsmentioning

confidence: 99%

“…Class I and Class II KARIs are also distinguished by the length of the Rossmann fold's β2αB-loop, which binds the adenine ribose of NAD(P)(H) and has been shown to be the primary determinant of whether an enzyme prefers NADH or NADPH [2, 21]. From a set of 558 Class II KARI sequences downloaded from Pfam [22], all but 20 have a 12-residue β2αB-loop.…”

Section: Introductionmentioning

confidence: 99%

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Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

Cahn

Brinkmann‐Chen

Spatzal

et al. 2015

Biochemical Journal

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Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are Class I enzymes, structural studies to date have focused primarily on the Class II KARIs, which arose through domain duplication. Here, we present five new crystal structures of Class I KARIs. These include the first structure of a KARI with a 6-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate binding geometry distinct from that of the 7- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in Class I KARIs upon binding of cofactor and metal ions. The Class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the Class II KARIs of rice and spinach and different from the opening of the active site observed in the Class II KARI of E. coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide binding site.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The tubes were incubated in their slots for 1 h, and then quenched on ice. Residual activity was determined with the activity assay described previously [2]. …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

Cahn

Brinkmann‐Chen

Spatzal

et al. 2015

Biochemical Journal

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Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

et al. 2019

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The asymmetric reduction of alkenes is a widely used transformation in industry. Ene reductases (ERs) are (βα) 8 -barrel folded enzymes capable of catalyzing this hydrogenation reaction. At the expense of nicotinamide coenzymes, ERs can reduce a wide range of electron-deficient alkenes in an anti-specific manner and with high regio-and stereoselectivities. However, a cost-effective industrial use of these enzymes is hampered, since most ERs prefer nicotinamide adenine dinucleotide phosphate (NADPH) to the more stable and less expensive non-phosphorylated nicotinamide adenine dinucleotide (NADH) as coenzyme. Here, we demonstrate an approach to both modify the biocatalysts coenzyme selectivity and strongly increase the activity and affinity with NADH. By swapping loop regions of the cyanobacterial NostocER1 for the corresponding regions of two NADH-favoring ERs, a strong alteration of the biocatalyst's coenzyme binding was achieved. This made possible a transfer of the respective donor-ER kinetic parameters to NostocER1. Additionally, outperformance of both donors in terms of activity was achieved through combinatorial swapping of loops of both species. These findings demonstrate the high potential of loop swapping as protein engineering approach to selectively optimize the coenzyme binding of ERs.

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Discovery of a Potentially New Subfamily of ELFV Dehydrogenases Effective for l‐Arginine Deamination by Enzyme Mining

Zhang

Huang

et al. 2017

Biotechnology Journal

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Discovery of enzymes with new functions is very important for de novo pathway design in synthetic biology. Amino acid dehydrogenases catalyze the oxidative deamination of an amino acid to its keto acid, which are widely used for the production of various valuable chemicals. To discover amino acid dehydrogenases with new functions, the authors reevaluate the sequence variability and substrate diversity of ELFV dehydrogenases superfamily in this study. With insights gained from structural and sequential studies, the authors develop an in silico strategy and discover a new category of proteins which are originally annotated as glutamate dehydrogenase but show altered conservation pattern of specificity determined motifs and completely different substrate spectrum. These proteins cannot catalyze the deamination of glutamate and other canonical amino acids except the positively charged amino acid l-arginine, representing a potentially new subfamily of ELFV dehydrogenases. The strategy utilized in this study can also be applied for discovering other useful enzymes.

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Uncovering rare NADH-preferring ketol-acid reductoisomerases

Cited by 27 publications

References 35 publications

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

Discovery of a Potentially New Subfamily of ELFV Dehydrogenases Effective for l‐Arginine Deamination by Enzyme Mining

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