Author's Accepted Manuscript
Uncovering rare NADH-preferring ketol-acid
reductoisomerases
S. Brinkmann-Chen, J.K.B. Cahn, F.H. Arnold
PII:
S1096-7176(14)00106-2
DOI:
http://dx.doi.org/10.1016/j.ymben.2014.08.003
Reference:
YMBEN917
To appear in:
Metabolic Engineering
Cite this article as: S. Brinkmann-Chen, J.K.B. Cahn, F.H. Arnold, Uncovering
rare NADH-preferring ketol-acid reductoisomerases,
Metabolic Engineering,
http:
//dx.doi.org/10.1016/j.ymben.2014.08.003
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Uncovering rare NADH-preferring ketol-acid reductoisomerases
Brinkmann-Chen, S.*; Cahn, J.K.B*; Arnold, F.H.
*These authors contributed equally
California Institute of Technology, Departmen
t of Chemistry and Chemical Engineering
Corresponding author: F. H. Arnold,
California Institute of Technology, 1
200 E California Blvd, MC 210-
41, Pasadena, CA 91125, USA
Phone: +1 626 395 4162
Email: frances@cheme.caltech.edu
sabine@cheme.caltech.edu
jcahn@caltech.edu
Abstract
All members of the ketol-acid reductoisomerase (KARI) enzyme family characterized to
date have been shown to prefer the nicotinamide adenine dinucleotide phosphate hydride
(NADPH) cofactor to nicotinamide adenine dinucleotide hydride (NADH). However,
KARIs with the reversed cofactor preference are desirable for industrial applications,
including anaerobic fermentation to produce branched-chain amino acids. By applying
insights gained from structural and engineering studies of this enzyme family to a
comprehensive multiple sequence alignment of KARIs, we identified putative NADH-
utilizing KARIs and characterized eight whose catalytic efficiencies using NADH were
equal to or greater than NADPH. These are the first naturally NADH-preferring KARIs
reported and demonstrate that this property has evolved independently multiple times,
using strategies unlike those used previously in the laboratory to engineer a KARI
cofactor switch.
Keywords: Ketol acid reductoisomerase; cofactor specificity; NADH; NADPH
1 Introduction
With burgeoning genomic databases and increasing ease of gene synthesis, metabolic
engineers can now readily mine nature’s rich collection of enzymes. However, finding a
sequence with specific desired properties can be difficult, particularly when only a few
members of a protein family have been characterized and a detailed understanding of the
structure-function relationship is lacking. The ketol-acid reductoisomerase (KARI, EC
1.1.1.86, also known as acetohydroxyacid isomeroreductase (AHAIR)) enzymes have
attracted much interest for production of amino acids and biofuels
(Atsumi et al., 2008a;
Bastian et al., 2011; Brinkmann-Chen et al., 2013; Hasegawa et al., 2012; Liu et al.,
2010). These oxidoreductases catalyze the second step in the branched chain amino-acid
(BCAA) biosynthesis pathway
(Chunduru et al., 1989), conversion of (
S
)-2-acetolactate
(
S
2AL) to (
R
)-2,3-dihydroxyisovalerate (
R
DHIV) via a methyl shift coupled to a
reduction with concomitant oxidation of a nicotinamide adenine dinucleotide cofactor.
The BCAA pathway is present in many organisms but not in mammals. Because of this,
microbial production of branched-chain amino acids for animal feed or human
supplements is a multimillion-dollar business
(Becker and Wittmann, 2012; Vogt et al.,
2014). The BCAA pathway has also been engineered to produce isobutanol, a potential
source of renewable chemicals and fuels
(Atsumi et al., 2008b).
All wild-type KARIs characterized and described in the literature have displayed a strong
preference for nicotinamide adenine dinucleotide phosphate (NADPH) over nicotinamide
adenine dinucleotide (NADH)
(Bastian et al., 2011). Because intracellular levels of
NAD(H) are much higher than NADP(H), particularly under fermentative conditions,
NADH-dependent oxidoreductases are strongly preferred in pathways for large-scale
biocatalytic processes
(Bastian et al., 2011). In the engineered isobutanol production
pathway, replacement of the natural
E. coli
KARI (Ec_IlvC) and the alcohol
dehydrogenase (ADH) with NADH-preferring engineered proteins increased the yield to
nearly 100% of theoretical and improved titer and specific productivity (Bastian et al.,
2011).
In a previous study that aimed to develop a general recipe for engineering KARIs with
reversed cofactor specificity, we used available KARI structure data to identify the amino
acid residues in the
2
B-loop of the Rossmann fold that distinguish between the two
cofactors
(Brinkmann-Chen et al., 2013). A sequence alignment of Swiss-Prot-annotated
KARI sequences allowed us to divide this diverse enzyme family into three groups based
on
2
B-loop length (6-, 7-, and 12-residue loops) and to develop a simple recipe for
switching the cofactor specificity of each major KARI enzyme subfamily from NADPH
to NADH. This engineering work provided valuable information on the determinants of
cofactor binding and also led us to question whether nature might have already
undertaken a similar engineering task to create an NADH-preferring KARI. Despite
recent advances in bioinformatic cofactor specificity prediction
(Geertz-Hansen et al.,
2014), few attempts have been made to find alternate cofactor utilization profiles within
large enzyme families
(Di Luccio et al., 2006). Because no method existed to predict the
cofactor specificity of uncharacterized KARIs based on their primary sequences, we used
knowledge gained from our previous work to exhaustively search known KARI
sequences for KARIs with
2
B-loops predicted to improve utilization of NADH. Here,