Tanja Knaus

Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades

A clean and green approach to amines Enzymes evolved to operate in water and to modify their substrates using comparatively nontoxic reagents. Thus, a major advantage of applying enzymes to synthetic chemistry is their compatibility with environmentally benign conditions. Mutti et al. report that two enzymes—alcohol and amine dehydrogenases—can operate in tandem to convert alcohols to amines. The reaction proceeds with ammonium as the only input and water as the only byproduct. The mechanism relies on consecutive oxidation and reduction steps, with hydrogen shuttled by a nicotinamide coenzyme. Science, this issue p. 1525 The pairing of two enzymes offers an environmentally benign protocol for the conversion of alcohols to amines. α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product.

Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes

The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the “ene” reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. “Better-than-Nature” biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost.

A Stereoselective Inverting sec-Alkylsulfatase for the Deracemization of sec-Alcohols

A metallo-β-lactamase-type alkylsulfatase was found to catalyze the enantioselective hydrolysis of sec-alkylsulfates with strict inversion of configuration. This catalytic event, which does not have an analog in chemocatalysis, yields homochiral (S)-configurated alcohols and nonreacted sulfate esters. The latter could be converted into (S)-sec-alcohols as the sole product in up to >99% ee via a chemoenzymatic deracemization protocol on a preparative scale.

One-pot deracemization of sec-alcohols: enantioconvergent enzymatic hydrolysis of alkyl sulfates using stereocomplementary sulfatases.

Given the fact that the theoretically possible number of racemates is larger than that of symmetric prochiral or meso compounds, the development of deracemization methods, which yield a single stereoisomer from a racemate is an important topic. Enantioconvergent processes are based on the transformation of a pair of enantiomers through opposite stereochemical pathways affecting retention and inversion of configuration. Depending on the stereochemical course of enzymatic and chemical reactions, three types of deracemization protocols were recently classified by Feringa et al. Two chemoenzymatic methods start with a biocatalytic kinetic resolution step, which yields a heteroor homochiral 1:1 mixture of the formed product and nonconverted substrate enantiomer. The latter is subjected to a second (nonenzymatic) transformation with retention or inversion of configuration to yield a single stereoisomeric product. Although several one-pot, two-step protocols have been successfully demonstrated, they typically rely on activated species, such as sulfonates, nitrate esters, or Mitsunobu intermediates, and negatively affect the overall atom economy of the process. The most elegant method relies on one (or two) enzyme(s), which mediate the transformation of both enantiomers through stereocomplementary pathways by retention and inversion. Since the requirements of such double selectivities are very difficult to meet, successful examples are rare: This approach has been applied to the hydrolysis of epoxides using two epoxide hydrolases showing opposite enantiopreference or a single enzyme that catalyzes the enantioconvergent hydrolysis of enantiomers with opposite regioselectivity. For enzymes, the ability to act by retention or inversion is a rare feature, which has been found among epoxide hydrolases, dehalogenases, 9] and sulfatases. The latter catalyze the hydrolytic cleavage of (alkyl) sulfate esters by breakage of the S O or the C O bond leading to retention or inversion at the chiral carbon atom, and thus makes them prime candidates for enantioconvergent processes. So far, only a single inverting sec-alkylsulfatase (PISA1) was generated recombinantly and characterized biochemically, thus allowing preparative-scale applications. In combination with acid-catalyzed hydrolysis of the nonreacted substrate enantiomer under retention of configuration a chemoenzymatic two-step deracemization protocol for sec-alcohols was recently developed. However, the method suffers from serious limitations because it requires undesirably large volumes organic solvents and several molar equivalents of a strong acid (typically 2–7 equiv of p-TosOH), which pose the risk of racemization or decomposition to the functionalized substrates, especially when elevated temperatures are required for acidic hydrolysis. Moreover, it is not applicable to retaining sulfatases, because no chemical method for sulfate ester hydrolysis with inversion exists. So far, retaining-sulfatase activity was reported in whole cells of Rhodopirellula baltica DSM 10527, but the corresponding enzymes could not be identified, thus impeding the use of recombinant technology to make the enzyme available for biocatalysis. Furthermore, the retaining sulfatase of Rh. baltica would not be suitable for an enantioconvergent process with PISA1, because both proteins exhibit the same enantiopreference. During our search for a retaining secalkylsulfatase with an enantiopreference opposite to that of PISA1, we discovered that the arylsulfatase from Pseudomonas aeruginosa (PAS) exhibited activity on sec-alkylsulfates. PAS, which has been characterized on a molecular level, showed promiscuous activity on various arylic phosphates and phosphonates. On its standard model substrate (4-nitrophenyl sulfate), PAS exhibited a rate acceleration of kcat/kuncat 2.3 10, and for a less reactive substrate the highest rate enhancement (kcat/kuncat = 2 10 ) of any catalytic reaction known so far has been measured. The stereochemical [*] M. Schober, M. Toesch, Dr. M. Fuchs, Prof. K. Faber Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz Heinrichstrasse 28, 8010 Graz (Austria) E-mail: kurt.faber@uni-graz.at Homepage: http://biocatalysis.uni-graz.at/

Alternative Hydride Sources for Ene‐Reductases: Current Trends

Asymmetric hydrogenations are important reactions in industrial synthesis, as up to two stereogenic centres can be generated. Given the current trend towards more “green” or sustainable chemistry, biocatalytic approaches are becoming more important in the production of fine chemicals, pharmaceuticals, and agrochemical products. There are at least four known classes of enzymes that have been explored for their biocatalytic applicability, collectively known as ene-reductases (ERs). Each enzyme requires NAD(P)H as the hydride donor, whereas the mechanism, substrate scope and product stereoand/or enantiospecificity differs between biocatalyst classes. The most widely investigated class is the old yellow enzyme (OYE; EC 1.6.99.1) family and many recent reviews have summarised the (potential) applications of these enzymes for industrial syntheses, for example, Refs. [1–3] . These flavin mononucleotide (FMN)-containing enzymes catalyse the C=C reduction of a wide variety of a,b-unsaturated aldehydes, ketones, nitroalkenes, maleimides, dicarboxylic acids and their esters, and nitriles. A second less well-characterised enzyme class catalysing biocatalytic reductions are enoate reductases (EC 1.3.1.31). These oxygen-sensitive enzymes contain both FAD and [4Fe-4S], and are members of the NADH:flavin oxidoreductase/NADH oxidase family. They have been shown to catalyse the reduction of a variety of a,b-unsaturated monoacids and esters. 5] A more recent class of enzymes investigated are the medium-chain dehydrogenase/reductase (MDR) family of oxidoreductases (1.3.1.-). For example, the flavin-independent double-bond reductase from Nicotiana tabacum (NtDBR) catalyses the reduction of a wide variety of a,b-unsaturated aldehydes, ketones and nitroalkenes. There are a few examples in the literature of potential applications of a fourth class of enzymes, namely the flavin-independent short-chain dehydrogenase/reductases (SDR) from plants. Examples include two menthol dehydrogenases, ( )-menthol dehydrogenase (EC 1.1.1.207) and (+)-neomenthol dehydrogenase (EC 1.1.1.208), which catalyse the reduction of specific menthone isomers to menthol. Unfortunately, the high cost of NAD(P)H nicotinamide coenzymes makes them uneconomical for industrial-scale syntheses. Therefore, alternative methodologies have been adopted to supply the necessary hydride equivalents for alkene reduction. One such method is to include a coenzyme recycling system, in which catalytic levels of NAD(P) are constantly regenerated to NAD(P)H. Several systems have been developed and routinely employed for ER biocatalytic reactions, such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH/glucose-6-phosphate), formate dehydrogenase and phosphite dehydrogenase, and reviewed elsewhere. In each case, only catalytic levels of NAD(P) are needed but stoichiometric levels of a co-substrate are required to drive the recycling enzyme. Faber and coworkers have performed comparative biotransformations by using multiple cofactor recycling systems, substrates and ERs, to determine the ideal hydride source. Product yield and/or enantioselectivity can vary considerably, depending on which coenzyme recycling system has been employed. Additionally, the inclusion of a second enzyme system into large-scale (bio)synthesis may present problems, for example in maintenance of the activity and stability of two enzymes and the high cost of some co-substrates. Electrochemical regeneration of nicotinamide coenzymes has also been investigated, for example in the direct cathodic reduction of NAD(P) . Unfortunately, this simple regeneration method is hampered by low selectivity and the formation of undesired NAD(P) dimeric side products. Several techniques have been developed to bypass the need for nicotinamide coenzymes by using alternative hydride donors to reduce the flavin cofactor of some ERs. Reetz and coworkers described a method whereby the FMN cofactor of the OYE homologue YqjM from Bacillus subtilis was photoreduced, employing free flavin and a sacrificial electron donor. The YqjM bound oxidised FMN was reduced by the free photoreduced FMN, thereby generating the active enzyme. This NAD(P)H-free system was successful in converting ketoisophorone to (R)-levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co-workers described the photoenzymatic flavin reduction of YqjM and NEMR (N-ethylmalemide reductase from E. coli) by using alternative sacrificial electron donors formate and phosphite. These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen. Early attempts at NAD(P)H-independent alkene reduction focussed on the use of artificial mediators, such as N,N-dimethyl4-4-bipyridinium [methyl viologen (MV)] , as a means of reducing the flavin in clostridial enoate reductases. A variety of reduced mediator recycling systems were employed, including direct electron transfer from a cathode and enzymatic-based recycling systems. Successful enzyme-coupled mediator recycling was achieved used the following systems: 1) hydroge[a] Dr. H. S. Toogood, Dr. T. Knaus, Prof. N. S. Scrutton Manchester Institute of Biotechnology, Faculty of Life Sciences University of Manchester 131 Princess Street, Manchester M1 7DN (UK) Fax: (+44)01613068918 E-mail : nigel.scrutton@manchester.ac.uk

Structure and mechanism of an inverting alkylsulfatase from Pseudomonas sp. DSM6611 specific for secondary alkyl sulfates

A highly enantioselective and stereoselective secondary alkylsulfatase from Pseudomonas sp. DSM6611 (Pisa1) was heterologously expressed in Escherichia coli BL21, and purified to homogeneity for kinetic and structural studies. Structure determination of Pisa1 by X‐ray crystallography showed that the protein belongs to the family of metallo‐β‐lactamases with a conserved binuclear Zn2+ cluster in the active site. In contrast to a closely related alkylsulfatase from Pseudomonas aeruginosa (SdsA1), Pisa1 showed a preference for secondary rather than primary alkyl sulfates, and enantioselectively hydrolyzed the (R)‐enantiomer of rac‐2‐octyl sulfate, yielding (S)‐2‐octanol with inversion of absolute configuration as a result of C–O bond cleavage. In order to elucidate the mechanism of inverting sulfate ester hydrolysis, for which no counterpart in chemical catalysis exists, we designed variants of Pisa1 guided by three‐dimensional structure and docking experiments. In the course of these studies, we identified an invariant histidine (His317) near the sulfate‐binding site as the general acid for crucial protonation of the sulfate leaving group. Additionally, amino acid replacements in the alkyl chain‐binding pocket generated an enzyme variant that lost its stereoselectivity towards rac‐2‐octyl sulfate. These findings are discussed in light of the potential use of this enzyme family for applications in biocatalysis.

Determination of free and bound riboflavin in cow’s milk using a novel flavin-binding protein

A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151μg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods.

Reverse structural genomics: an unusual flavin-binding site in a putative protease from Bacteroides thetaiotaomicron.

Background: The structure of a putative protease from Bacteroides thetaiotaomicron has a unique binding site for a flavin. Results: The protein tightly binds to lumichrome, riboflavin, FMN, FAD, and flavin derivatives. Conclusion: The putative protease is a scavenger of flavin and may function as a storage protein in gut bacteria. Significance: The wealth of information available for cofactors is invaluable to assess protein structures. The structure of a putative protease from Bacteroides thetaiotaomicron features an unprecedented binding site for flavin mononucleotide. The flavin isoalloxazine ring is sandwiched between two tryptophan residues in the interface of the dimeric protein. We characterized the recombinant protein with regard to its affinity for naturally occurring flavin derivatives and several chemically modified flavin analogs. Dissociation constants were determined by isothermal titration calorimetry. The protein has high affinity to naturally occurring flavin derivatives, such as riboflavin, FMN, and FAD, as well as lumichrome, a photodegradation product of flavins. Similarly, chemically modified flavin analogs showed high affinity to the protein in the nanomolar range. Replacement of the tryptophan by phenylalanine gave rise to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was abolished. We propose that the protein is an unspecific scavenger of flavin compounds and may serve as a storage protein in vivo.

Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron.

The crystal structure of a putative protease from Bacteroides thetaiotaomicron (ppBat) suggested the presence of a zinc ion in each protomer of the dimer as well as a flavin in the dimer interface. Since the chemical identity of the flavin and the exact mode of binding remained unclear, we have determined the crystal structure of ppBat in complex with riboflavin. The obtained structure revealed that the isoalloxazine ring is sandwiched between two tryptophan residues (Trp164) from both chains and adopts two alternate orientations with the N(10)-ribityl side chain protruding from the binding site in opposite directions. In order to characterize the zinc-binding site, we generated two single variants and one double variant in which the two coordinating cysteine residues (Cys74 and Cys111) were replaced by alanine. All three variants were unable to bind zinc demonstrating that both cysteine residues are essential for binding. Moreover, the lack of zinc binding also resulted in drastically reduced thermal stability (11-15°C). A similar effect was obtained when wild-type protein was incubated with EDTA supporting the conclusion that the zinc-binding site plays an important structural role in ppBat. On the other hand, attempts to identify proteolytic activity failed suggesting that the zinc may not act as a catalytic center in ppBat. Structurally similar zinc binding motives in other proteins were also found to play a structural rather than catalytic role and hence it appears that neither the flavin nor the zinc binding sites possess a catalytic function in ppBat.