Stereoselective nitrile hydratases

Wu, S., Fallon, R.D. and Payne, M.S. (1997) Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Pichia pastoris activity requires a novel downstream protein. Applied Microbiology and Biotechnology, 48 (6), 704—708. 

In a number of gram-positive organisms the combination of a stereoselective nitrile hydratase and a stereoselective amidase has been described (see chapter below). However, in the case of the acetonitrile-utilizing Rhodococcus sp. AJ 270 no amidase activity has been detected [55]. The wide-spectrum nitrile hydratase of this microorganism was used to prepare (/ )-2-phenylbutyramide from the racemic nitrile without acid as byproduct (Fig, 14). 

V. STEREOSELECTIVE NITRILE HYDRATASES IN COMBINATION WITH STEREOSELECTIVE AMIDASES... 

Among stereoselective nitrile-converting enzymes, the combined action of a stereoselective nitrile hydratase and a stereoselective amidase has been often described, and stereospecific nitrile conversions by whole cells are frequently found in the patent literature [59,60] however, careful analysis has usually revealed stereoselectivity in the amidase and not or only to a low extent in the hydratase [61,62]. If both enzymes were stereosj ific, nitrile hydratase and amidase have been described to act either synergistically or antagonistically regarding their enantioselectivity. 

Figure 17 Racemic resolution of (/ ,5)-2-arylpropionitriles by stereoselective nitrile hydratase/ amidase systems from Rhodococcus sp. SP 361 and Agrobacterium tumefaciens d3.

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

The ease of the Strecker synthesis from aldehydes makes a-aminonitriles an attractive and important route to a-amino acids. Fortunately, the microbial world offers a number of enzymes for carrying out the necessary conversions, some of them highly stereoselective. Nitrilases catalyze a direct conversion of nitrile into carboxylic acid (Equation (11)), whereas nitrile hydratases catalyze formation of the amide, which can then be hydrolyzed to the carboxylic acid in a second step (Equation (12)). In a recent survey, with a view to bioremediation and synthesis, Brady et al have surveyed the ability of a wide range of bacteria and yeasts to grow on diverse nitriles and amides as sole nitrogen source. This provides a rich source of information on enzymes for future application. 

Although nitrile hydratases tend not to be stereoselective, examples of enantioselective enzymes are known [103, 106, 107, 114]. Of particular interest is the possibility to selectively hydrolyse 2-phenylproprionitriles, the core structure for ibuprofen and many other profens [103, 107, 114, 115]. This enables the enantioselective synthesis of the amides of ketoprofen and naproxen (Scheme 6.39). 

Strategic importance of biocatalyzed synthetic transformations in terms of eco-compatibility and cheaper processes has been widely stressed previously. Among the developed biotransformations catalyzed by nitrilases or nitrile hydratases/ amidases systems, a special interest is focused toward stereoselective reactions able to give access to molecules otherwise impossible to obtain by classical chemical routes. Hereby, selected examples aim to offer an overview of research in this direction. Examples of industrial processes using nitrile hydrolyzing biocatalysts are also illustrated. 

Despite the fact that early experiments suggested low selectivity of nitrile-converting enzymes with respect to the substrate chirality (Faber, 1992), many recent works report the successful enantioselective bioconversion of nitriles catalyzed by nitrilases or nitrile hydratases, even if the stereoselectivity of nitrile hydratases remains often lower that that of coupled amidases. 

Nitrile Hydratase/Amidase Catalyzed Stereoselective Transformation of cis- and frans-N-Protected-P-amino-cyclopentane/ hexane Nitriles... 

During our longstanding interest in the biohydrolysis of nitriles, we found that whole cell preparations of certain Rhodococci, such as R. erythropolis A4 (formerly R. equi A4), R. sp. R312, and R. erythropolis NCIMB 11540, containing the nitrile hydratase/amidase enzyme system, are efficient catalysts for stereoselective microbial hydrolysis of N-protected carbocyclic P-amino nitriles ( )-la-( )-4a, to P-amino acids lc-4c and amides lb-4b, respectively (Scheme 15.1) [33, 34]. 

So far from the numerous results achieved with a variety of strains generated in different culture conditions a broad substrate tolerance or stereoselectivity of the enzyme has emerged, even though interpertation of the results has been rather compHcated. Nitrile hydratases are considered to be active preferentially on aliphatic nitriles with only a marginal activity on aromatic ones [14, 15, 24—26]. 

The degradation of nitriles by nitrilases (EC 3.5.5.1) has been the subject of intense study, especially as it relates to the preparation of the commodity chemical acrylamide. Nitrilases catalyze the hydrolysis of nitriles to the corresponding acid plus ammonia (Figure 1 reaction 5), whereas nitrile hydratases (EC 4.2.1.84) add water to form the amide. Strains such as Rhodococcus rhodo-chrous Jl, Brevibacterium sp., and Pseudomonas chlororaphis have been used to prepare acrylamide from acrylonitrile, which contain the hydratase and not nitrilase activity [12]. A comparison of these strains has been discussed elsewhere [98]. Other uses of nitrilases, however, have primarily been directed at resolution processes to stereoselectively hydrolyze one enantiomer over another or regiose-lectively hydrolyze dinitriles [99-101]. 

A biocatalytic process was recently disclosed in which the strategic step involves kinetic resolution of a racemic 2-pyrrolidinonyl nitrile by nitrile hydratases (Scheme 8.15) [65, 66]. Using an engineered nitrile hydratase mutant, the enzymatic step proceeds with high productivity (100 g l-1 per day), good resolution yield (43%), and high stereoselectivity (94% ee). The biocatalytic process can potentially be much... 

Types of selectivity exhibited by enzymes. Biocatalytic processes can provide chemo-, regio-, and stereoselective conversions. Representative examples can be observed in reactions catalyzed by nitrile hydratases, lipases and dehydrogenases. 

Previous reviews of this field of research focused mainly on the biocatal5dic uses of nitrilases [1], structure and function of nitrilases [2, 3], nitrilases in filamentous fungi [4], stereoselective biotransformations catalyzed by nitrile-converting enz5nnes [5], and the structure, function, and uses of nitrile hydratases [6]. For a comprehensive review of nitrile-converting enzymes known at the time, the study by Banerjee et al. [7] has been helpful. Some of the most recent reviews focused on nitrilases, specifically their sources, properties, and use, [8], and on methodologies for their screening [9]. 

Nitrilases catalyze the direct hydrolysis of nitriles to the corresponding acids. Compared to nitrile hydratases, more nitrilases bearing a high stereoselectivity have been described. Nitrilases generally comprise of one type of subunit in multiple association (ae-ao) with a few monomeric (a) and dimeric (aj) exceptions. As a rough rule, they are mostly active with aromatic nitriles but exhibit high activity toward aliphatic nitriles less often [20-24]. 

The application of the immobilized nitrilase SP 409 of Rhodococcus sp. from Novo Industri (Denmark), which covers a wide substrate spectrum of aliphatic, aromatic, het-erocylic, and carbohydrate nitriles, proved to be synthetically viable for the mild and stereoselective transformation of base-sensitive carbohydrate nitriles [39]. Preferentially the P anomer of a diastereomeric glycosyl cyanide was hydrolyzed to the corresponding acid (Fig. 8). Using a C-7 alkoxylated glycosyl cyanide, amide intermediates were also detected, indicating the additional presence of a nitrile hydratase. 

Evidence for the enantioselectivity of the nitrile hydratase was given by the purified enzyme catalyzing the hydration of the (5)-nitrile at least 50 times faster than the hydrolysis of the (i )-nitrile [51]. The strain was also capable of a two-step hydrolysis of racemic ibuprofen and naproxen nitriles to the corresponding (S)-acids in enantiomeric purities above 90% e.e., however, with the stereoselectivity residing primarily in the amidase. In this case, the analysis of enantioselectivity was complicated due to the product inhibition of (i )-nitrile hydration by enzymatically formed (5)-amide. On the basis of the initial rate of appearance, the nitrile hydratase showed a slight preference for the (R) enantiomers of... 

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