Genetic and Functional Diversity of Nitrilases in Agaricomycotina

Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, β-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.


Supplementary data -Nitrilase sequences in Agaricomycotina
The enzymes overproduced in E. coli are marked in bold. NitAg, NitSh and NitTv1 were produced in this study, NitAd in the previous study (ref. [16] in the main manuscript). Proteins with 99% amino acid sequence identity, which occur in the same species, were discarded.
Sequences in blue do not belong to clade 1 or 2 or CynHs.

Products of fumaronitrile transformations by nitrilase NitTv1
To  To obtain products for NMR (Figs S4 and S5), the conditions of the transformation of FN were modified (dry cell weight 0.6 g/L, reaction time 60 min, total volume 50 mL).
After removing the cells by centrifugation, the supernatant was extracted with ethylacetate at pH 8 (pH of the reaction mixture) and then at pH 2 (adjusted with 2M HCl). The organic fractions from each extraction were pooled, dried with Na2SO4 and filtered, and the solvent was removed at reduced pressure.

Products of fumaronitrile transformations by nitrilase NitAg
To obtain samples for LC-MS from the reaction using whole cells carrying NLase NitAg, transformation of FN was carried out analogously as described for NitTv1 above but with modifications (dry cell weight 4.5 g/L, reaction time 120 min, total volume 0.5 ml). The supernatant was diluted with mobile phase (1:50) and analyzed by LC-MS (see Fig. S6 for the chromatogram and figure legend for m/z (ESI) data).

Products of fumaronitrile transformations by nitrilase NitSh
Transformation of FN by NitSh was carried out analogously as described for NitTv1 above but with modifications (dry cell weight 3 g/L, reaction time 60 min and total volume 50 mL). The products were isolated in the same way as in the previous experiment. The product extracted at pH 8 (27 mg) contained a mixture of the residual substrate 1a and product 1c at a ratio of ca. 2 : 3. The product extracted at pH 2.5 contained compound 1b as the major product (isolated yield 69%; 41 mg). The products were analyzed by NMR (Figs S7 and S8).

Product of 3-phenylpropionitrile transformation by nitrilase NitTv1
The transformation of 3-phenylpropionitrile (PPN) by NitTv1 was carried out analogously as described for FN above but with modifications (dry cell weight 3 g/L, reaction time 120 min, total volume 0.5 mL). The sample for LC-MS was prepared as described for FN above (see Fig. S9 for the chromatogram and figure legend for m/z (ESI) data).

Products of -cyanoalanine transformation by nitrilase NitTv1
The transformation of -cyanoalanine (-CA) by NLase NitTv1 was carried out analogously as described for FN but with modifications (dry cell weight 0.6 g/L, reaction time 60 min, total volume 50 mL). The supernatant was lyophilized and analyzed by NMR (Figs S10 and S11).

Products of 2-cyanopyridine transformation by nitrilase NitSh
The transformation of 2-cyanopyridine (2CP) by NitSh was carried out analogously as described for FN above but with modifications (dry cell weight 0.3 g/L, reaction time 10 min, total volume 0.5 ml). The samples for LC-MS were prepared as described for FN above (see Fig. S12 for the chromatogram and figure legend for m/z (ESI) data).

Products of 4-cyanopyridine transformation by nitrilase NitTv1
The transformation of 4-cyanopyridine (4CP) by NitTv1 was carried out analogously as described for FN above but with modifications (dry cell weight 3 g/L, reaction time 120 min). The samples for LC-MS were prepared as described for FN above (see Fig. S13 for the chromatogram and figure legend for m/z (ESI) data).

Products of benzonitrile transformation by nitrilase NitSh
The transformation of benzonitrile (BN) by NitSh was carried out as described for FN above but with modifications (dry cell weight 0.3 g/L, reaction time of 5 min, total volume 0.5 ml). The product (benzoic acid) was determined by HPLC as described in Materials and methods and its UV spectrum was compared with that of the authentic standards (absorption maximum at 228.7). No significant amount of benzamide (absorption maximum at 225.2 nm) was found in the reaction mixture