Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from Pseudomonas fluorescens EBC191

Abstract

The arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse (R,S)-mandelonitrile to (R)-mandelic acid with an enantiomeric excess (ee) of 91% or to (S)-mandelic acid with an ee-value of 47%. The conversion of (R,S)-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either (S)- or (R)-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile could be changed by single point mutations between 2%–94% and <0.2%–73%, respectively. The present study attempted to collect and compare the results obtained during our previous work, and to obtain additional general information about the relationship of the amide forming capacity of nitrilases and the enantiomeric composition of the products.

Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764

“…Each commercial compound yielded a similar pair of doublets with chemical shift values in line with those of the biologically-formed product but with distinctly different coupling constants; 15.6 for the trans versus 12.4 for the cis species (data not shown). Accordingly, the higher value of 15.6 Hz for the bioconversion product gave reason to conclude that the trans isomer of the monoamide ( trans -3-cyanoacrylamide) was formed and is consistent with the conventional mechanism of nitrilase-catalyzed reactions in which small quantities of the amide intermediate can escape from the enzyme [ 1 , 2 , 36 ].

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“…The demonstrated broad specificity for relatively large nitrile molecules is consistent with this. Moreover, like Nit6803 and other nitrilases capable of attacking dinitriles [ [31] , [32] , [33] , [34] , [35] , [36] ], Nit11764 also is able to act on only one of the cyano groups, the monocarboxylate being formed identified by 1 HNMR as trans -3-cyanoacrylate ( Fig. 6 ).…”

Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase

“…The analysis of the mandelic acid formed by chiral HPLC demonstrated that in both systems almost exclusively (R)-mandelic acid was formed (ee > 95%). This clearly demonstrated that the ( R )-oxynitrilase and the nitrilase were active in the whole cell catalysts because it was previously shown that the nitrilase from P. fluorescens EBC191 converts mandelonitrile in the absence of an oxynitrilase activity only with much lower enantioselectivity to ( R )-mandelic acid (with an ee of about 30%) and additionally forms higher amounts of mandelic acid amide from racemic mandelonitrile (Kiziak et al 2007 ; Stolz et al 2019 ).…”

“…Furthermore, (substituted) mandelic acid amide(s) are building blocks of several antibiotics, fungicides, and antioxidatives (Cederbaum et al 2004 ; Cole 1969 ; Ley and Bertram 2001 ). Therefore, it was disappointing that all previously studied variants of the nitrilase from P. fluorescens EBC191 which formed significant amounts of mandelic acid amide only formed ( S )-mandelic acid amide with a rather low-degree of enantioselectivity (Stolz et al 2019 ). In contrast, the efficient synthesis of ( R )-mandelic acid amide shown in the present study by the system expressing the ( R )-specific oxynitrilase and the amide forming nitrilase variant, opens new perspectives for the enantioselective synthesis of ( R )-hydroxycarboxamides.…”

Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis

“…Although molecular modeling of nitrilases and the interactions between their subunits and substrates have been reported [ 15 , 16 , 17 , 18 ], details regarding the molecular determinants of the catalytic efficiency of arylacetonitrilase have never, to the best of our knowledge, been reported. An understanding of the role of amino acid residues at the active microenvironment site may guide future protein design efforts to exploit the ubiquitous and industrially useful enzymes such as nitrilases [ 19 ].…”