Bettina M. Nestl

New generation of biocatalysts for organic synthesis.

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts--reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases--will be discussed herein and exemplified by the syntheses of interesting compounds.

Recent progress in industrial biocatalysis

In recent years, several procedures have been reported for the development of biocatalytic processes. This review focuses on selected examples integrating biocatalysts into a variety of industrially interesting processes ranging from the manufacture of smaller, chiral speciality chemicals to the synthesis of more complex pharmaceutical intermediates. The use of rational protein design, multistep processes and de novo design of enzyme catalysts for the stereocontrolled preparation of important target structures is discussed.

Synthesis of ω‐hydroxy dodecanoic acid based on an engineered CYP153A fusion construct

A bacterial P450 monooxygenase‐based whole cell biocatalyst using Escherichia coli has been applied in the production of ω‐hydroxy dodecanoic acid from dodecanoic acid (C12‐FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq.‐CPR) was used for the hydroxylation of C12‐FA in in vivo studies. These experiments yielded 1.2 g l–1 ω‐hydroxy dodecanoic from 10 g l–1 C12‐FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12‐FA methyl ester as substrate in a two‐phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l–1 ω‐hydroxy dodecanoic acid. We further summarized the most important aspects of the whole‐cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

Enzyme Toolbox: Novel Enantiocomplementary Imine Reductases

Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine‐chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of CO and activated CC bonds. However, stereoselective enzymatic reduction of CN bonds is still in its infancy—it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine‐reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2‐methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2‐methylpyrroline in whole cells with excellent selectivities.

Bacterial CYP153A monooxygenases for the synthesis of omega-hydroxylated fatty acids

CYP153A from Marinobacter aquaeolei has been identified as a fatty acid ω-hydroxylase with a broad substrate range. Two hotspots predicted to influence substrate specificity and selectivity were exchanged. Mutant G307A is 2- to 20-fold more active towards fatty acids than the wild-type. Residue L354 is determinant for the enzyme ω-regioselectivity.

Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666

The oxofunctionalization of saturated hydrocarbons is an important goal in basic and applied chemistry. Biocatalysts like cytochrome P450 enzymes can introduce oxygen into a wide variety of molecules in a very selective manner, which can be used for the synthesis of fine and bulk chemicals. Cytochrome P450 enzymes from the CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. Here we report the product yields resulting from C(5)-C(12) alkane and alcohol oxidation catalyzed by CYP153A enzymes from Mycobacterium marinum (CYP153A16) and Polaromonas sp. (CYP153A P. sp.). For all reactions, byproduct formation is described in detail. Following cloning and expression in Escherichia coli, the activity of the purified monooxygenases was reconstituted with putidaredoxin (CamA) and putidaredoxin reductase (CamB). Although both enzyme systems yielded primary alcohols and α,ω-alkanediols, each one displayed a different oxidation pattern towards alkanes. For CYP153A P. sp. a predominant ω-hydroxylation activity was observed, while CYP153A16 possessed the ability to catalyze both ω-hydroxylation and α,ω-dihydroxylation reactions.

Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis

For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on natures catalytic machineries to generate unique catalysts.

Imine Reductase‐Catalyzed Intermolecular Reductive Amination of Aldehydes and Ketones

Imine reductases (IREDs) have emerged as promising biocatalysts for the synthesis of chiral amines. In this study, the asymmetric imine reductase‐catalyzed intermolecular reductive amination with NADPH as the hydrogen source was investigated. A highly chemo‐ and stereoselective imine reductase was applied for the reductive amination by using a panel of carbonyls with different amine nucleophiles. Primary and secondary amine products were generated in moderate to high yields with high enantiomeric excess values. The formation of the imine intermediate was studied between carbonyl substrates and methylamine in aqueous solution in the pH range of 4.0 to 9.0 by 1H NMR spectroscopy. We further measured the kinetics of the reductive amination of benzaldehyde with methylamine. This imine reductase‐catalyzed approach constitutes a powerful and direct method for the synthesis of valuable amines under mild reaction conditions.

Variations in the stability of NCR ene reductase by rational enzyme loop modulation.

The engineering of protein stability is of major importance for the application of enzymes in a wide range of industrial applications. Here we study the determinants of the thermo- and solvent stability of the Zymomonas mobilis ene reductase NCR using a rational protein engineering approach based on analyses of structural and sequence data. We designed and created two loop mutants with the aim to increase their overall stability. They all retained catalytic activity but exhibited altered thermostability relative to the wild-type enzyme. The modulation of one specific loop segment near the active site of NCR showed an increased tolerance to organic solvents along with an enhanced thermostability.

Crystal structure determination and mutagenesis analysis of the ene reductase NCR.

The crystal structure of the “ene” nicotinamide‐dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β‐unsaturated alkenes, the rational design methods site‐ and loop‐directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single‐residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop‐swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild‐type NCR towards the set of substrates tested. The active‐site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop‐swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild‐type NCR ene reductase enzyme.