Identification of novel cold-adapted nitrilase superfamily enzymes
Nel, Andrew James Mascré
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In bacteria, nitrile hydratases and enzymes of nitrilase and signature amidase superfamilies hydrolyse nitriles and amides to their corresponding carboxylic acids releasing ammonia. Bacteria expressing these enzymes are typically isolated where a sole nitrogen and/or carbon source is used to support their growth. The majority of characterised enzymes of industrial potential have been identified for their stabilities at elevated temperatures. To date, no reports of such enzymes have been isolated from cold adapted bacteria.In this study, an extensive screening program of cold-active microbial isolates for enzymes of this group led to the selection and detailed characterisation of an aliphatic amidase from Nesterenkonia.Nesterenkonia AN1, a new psychrotrophic isolate of the genus, was isolated from soil samples collected from the Miers Valley, Antarctica. AN1 showed significant 16S rRNA sequence identity to known members of the genera, but this is the only strain that had optimal growth at approximately 21oC. AN1, similar to known members, is an obligately alkaliphilic (pH 9-10) and halotolerant (Na+ 0- 15% (w/v)) strain.The genome of Nesterenkonia AN1, sequenced in-house, revealed two ORFs encoding putative nitrilases, referred to as Nit1 and Nit2. Based on analysis of their deduced protein sequences, both belonged to the nitrilase superfamily. Both sequences showed conserved catalytic residues (EKEC), glycine residues and contained the characteristic áââá monomer fold. Homology modelling using known structures suggested that both genes could encode N-carbamoyl D-amino acid amidohydrolases, although neither showed conserved residues implicated in the hydrolysis of carbamoyls.Nit1 and Nit2 were expressed in Escherichia coli BL21 (DE3) pLysS as Cterminal and N-terminal hexahistidine tagged fusion proteins, and purified using Ni-chelation chromatography. Nit1 showed no activity towards nitrile, amide and carbamoyl substrates. This protein, unlike members of the multimeric enzymes of the nitrilase superfamily, was a monomer ~30 kDa protein. It is possible that the C-terminal hexahistidine tag might have prevented Nit1 from forming multimeric proteins.Nit2 showed substrate specificity similar to known aliphatic amidases with a preference for small amides. Nit2 had maximal activity at 30oC and between pH 6.5 and 7.5, properties compatible with its cold-adapted alkaliphilic origins. In addition, the enzyme was irreversibly inactivated at temperatures above 30oC and had a half-life of approximately 7 mins at 60oC. The crystal structure of Nit2 was solved to 1.66 Å. It revealed a ~45.5 kDa dimer, composed of two tightly bound ~30 kDa monomers. These monomers associated along the A surface forming a áââá-áââá sandwich architecture that is conserved in known structures of the nitrilase superfamily.Nit2 is distinct from known aliphatic amidases in both its structure and enzymic activity: the enzyme did not possess an extended C-terminal region; is active in dimeric form; has high affinity for 3C amides rather than 2C amides; and has a low overall catalytic rate. The short C-terminal region of Nit2 may have contributed to the low stability of the enzyme at elevated temperatures. A dendrogram composed of protein sequences of members of the nitrilase superfamily and Nit2 further supported evidence that this aliphatic amidase falls within a distinct group of enzymes.This is the first report of the enzymic characterisation and structural analysis of an aliphatic amidase from a psychrotolerant, alkaliphilic and halotolerant extremophile.