Nitrile hydratase substrates

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

Hensel et al. have also reported the use of a-amino nitriles as substrates (Scheme 2.10). They discovered a number of new bacterial isolates with stereose-lechve nitrile hydratase achvity. A combination of stereoselechve nitrile hydratases and amidases was shown to be responsible for the production of phenylglycine 24 from nitrile 22 via amide 23. By investigating five isolates both (R)- and (S)-phenylglycine were produced in greater than 99% e.e. [12]. 

There are two distinct classes of enzymes that hydrolyze nitriles. Nitrilases (EC. 3.5.5.1) hydrolyze nitriles directly to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nitriles also may be first hydrated by nitrile hydratases to yield amides which are then converted to carboxylic acid with amidases. This is u two-enzyme process, in which enanlioselectivity is generally exhibited by the amidase. rather than the hydratase. 

Cyanide hydratase and cyanide dihydratase belongs to the nitrilase branch of nitrilase superfamily, using HCN as the only efficient substrate and producing amide and acid products, respectively. Microorganisms appear in fact to have evolved separate metabolic pathways for the hydrolysis of inorganic cyanide. Thus, most nitrilases (as well as nitrile hydratases) till now investigated do not display activity... 

Organisms with Characterized Nitrile Hydratase Activity and Their Substrates... 

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. 

These data and considerations on structural features of substrates have suggested the hypothesis that a readily reachable reactive site is embedded within a spacious pocket of the enantioselective nitrile hydratase, while the amidase comprises a relatively deep-buried and size-limited enantioselective active site. This hypothesis has been supported by the results obtained studying the biotransformations of differently configured 2,2-dimethyl-3-arylcyclopropanecarbonitriles catalyzed by... 

Wieser, M., Takeuchi, K., Wada, Y. et al. 1998. Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous JI Purification, substrate specificity and comparison with the analogous high molecular-mass enzyme. FEMS Microbiology Letters, 169 17-22. 

However, the rise of more sophisticated analytical techniques has triggered communications on this subject substantially in recent times. This nitrile hydratase activity of nitrilases was frequently observed when substrates activated in the a-position to the nitrile group were used [45, 46]. Most recently, a study appeared on this subject suggesting a mechanistic rationale of amide formation [47]. 

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]. 

Moreover, nitrile hydratases have been found to be thermally unstable, substrate inhibited, and also inhibited by the amides or adds produced during bioconversion. 

The operational thermal stability of enzymes can be easily evaluated in experiments carried out in a CSMR fed with a saturating substrate concentration, while varying the temperature but maintaining all the other parameters constant. Each enzyme of the cascade system was tested by feeding the CSMR with the appropriate substrate. The kinetic characterization of amidase-catalyzed reactions in runs fed with a nitrile was hampered by the fact that the intracellular enzyme works in cascade with nitrile hydratase. The concentration of amide, produced in situ in the first step, varied with the time and reaction conditions and did not assure the differential conditions needed for an accurate analysis, the amide being completely converted by amidase in some runs. Hence, amidase activity was characterized independently by feeding the reactor with amide as the substrate [35]. 

Figure 173 (a) Time course of reaction rate in UF-membrane bioreactor at different temperature. Appropriate substrate feed solution (benzonitrile or benzamide) lOmM, resting cell load 2mgocw, flow-rate 12mlh. Filled symbols for nitrile hydratase activity ... 

Similar data were obtained for 3-cyanopyridine biotransformation into nicotinamide and nicotinic acid (unpublished data). The higher dependence of the nitrile hydratase deactivation process on temperature has already been observed with other substrates, such as in acrylonitrile bioconversion into acrylamide where the nitrile hydratase half-Ufe dropped from 33 h to approximately 7h when the temperature was varied from 4 to 10 °C [37]. 

During the course of our research on nitrile hydratase- and amidase-catalyzed reactions we had to deal with different aspects of substrate concentration effects on the enzyme kinetics and all were suitably investigated making use of a CSMR, as shown by the following case studies. 

The effect of the substrate (benzonitrile) concentration on nitrile hydratase activity was investigated in a CSMR in the range of 2-10mM at low temperature (10 °C) in order to reduce the enzyme thermal inactivation as much as possible. The experimental runs are illustrated in Figure 17.4a as a semi-logarithmic plot of the specific reaction rate against process time. Both the r - and the ka-values were calculated (as summarized in the previous paragraph) and are quoted in Table 17.1. 

The propionitrile nitrile hydratase-catalyzed reaction presented kinetics inhibited by high substrate concentrations that were clearly evidenced in the CSMR, as revealed in [33]. The kinetic parameters for nitrile hydratase-catalyzed reactions of... 

Figure 17.5 Scheme of the reaction runs performed in UF-membrane bioreactor in the presence of high substrate concentration inactivating the nitrile hydratase activity (for... 

This was performed for each enzyme independently, feeding the reactor with the appropriate substrate (nitrile for the cascade reaction, amide for the sole amidase). The activation energies of both catalysed reactions were evaluated together with those of the inactivation process that inevitably takes place even under the most suitable operational conditions. In the nitrile hydratase/amidase cascade system nitrile hydratase is the more labile enzyme that imposes process temperature choice. These findings make accessible the complete kinetic expression of the dependence from temperature of reaction rate, allowing accurate prediction on reactor performances for process scale-up. 

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