Acceleration of an aldo-keto reductase by minimal loop engineering

Krump, Corinna; Vogl, Michael; Brecker, Lothar; Nidetzky, Bernd; Kratzer, Regina

doi:10.1093/protein/gzu021

Cited by 6 publications

(4 citation statements)

References 15 publications

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“…The substrate-binding cavity of aldo-keto reductases is mainly formed by residues from three large and flexible loops [20,21]. Loop flexibility provides the structural basis for the relaxed substrate specificity, but complicates rational engineering [22].…”

Section: Ctxr Mutantsmentioning

confidence: 99%

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

et al. 2021

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Background Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase. Results A single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·103 s−1 M−1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee. Conclusion The current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.

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Section: Ctxr Mutantsmentioning

confidence: 99%

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

et al. 2021

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“…Previous studies on AKR superfamily members report successful efforts at engineering such variants [64][65][66]. For instance, in CtXR, a redesign of loop 1 that contains residue Trp19 (in DnXR), yielded a variant with enhanced turnover number and enantioselectivity for bulky ketones of industrial relevance [67].…”

Section: Structural Basis Of Substrate Recognitionmentioning

confidence: 99%

Crystal structure of yeast xylose reductase in complex with a novel NADP‐DTT adduct provides insights into substrate recognition and catalysis

2018

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Aldose reductases (ARs) belonging to the aldo‐keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP‐DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l‐threose (138 ± 7 mm−1·s−1) followed by d‐erythrose (30 ± 3 mm−1·s−1). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP–low‐molecular‐mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in‐crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four‐carbon sugar alcohol analog, and the enzyme are representative of a near‐cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. Database Structural data are available in the PDB under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCI and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCM.

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“…Meanwhile, substituting the bulky Gln to much smaller Ala also decreases the steric hindrance for NADPH binding. [ 44,45 ] (Figure S7). The remaining two changes of M5‐Q213A/T23V were located in the region of active center, one was Asp59 and the other was Pro254, where the secondary structure of β ‐sheet changed to random coil, enhancing flexibility, resulting in the increase of activity.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope

Qiu

et al. 2021

Biotechnology Journal

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Background Aldo‐keto reductases‐catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo‐keto reductase (AKR) from Kluyveromyces marxianus that is active to the aliphatic tert‐butyl 6‐substituted (5R/S)‐hydroxy‐3‐oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent KmAKRmutant for ensuring the simultaneous improvement of activity‐thermostability toward tert‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐1) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence‐based high‐throughput (HT) screening technique was established. Main Methods and Major Results The directed evolution of KmAKR variant M5 (KmAKR‐W297H/Y296W/K29H/Y28A/T63M) produced the “best” variant M5‐Q213A/T23V. It exhibited enhanced activity‐thermostability toward (5R)‐1, improved activity toward all 18 test substrates and strict R‐stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5‐Q213A/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94 g dry cell weight (DCW) L−1 glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration, 2.81 g DCW L−1 M5‐Q213A/T23V completely converted (5R)‐1 of up to 450 g L−1 at 120 g g–1 substrates/catalysts (S/C), yielding the corresponding optically pure tert‐butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐2, > 99.5% d.e.p) with a space‐time yield (STY) of 1.08 kg L−1 day−1. Conclusions A fluorescence‐based HT screening system was developed to tailor KmAKR's activity, thermostability and substrate scope. The “best” variant M5‐Q213A/T23V holds great potential application for the synthesis of aliphatic/aromatic R‐configuration alcohols.

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Acceleration of an aldo-keto reductase by minimal loop engineering

Cited by 6 publications

References 15 publications

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

Crystal structure of yeast xylose reductase in complex with a novel NADP‐DTT adduct provides insights into substrate recognition and catalysis

Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope

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