Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase.

Jason M. Park,B. Trevor Sewell,Andani Mulelu,Michael J. Benedik

doi:10.3389/fmicb.2015.01479

Jason M. Park, B. Trevor Sewell + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2015.01479

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Abstract

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases.

Highlights

Nitrilase enzymes from the large nitrilase-superfamily are of significant industrial interest due to their ability to process nitrile compounds into valuable acid products such as nicotinic acid, acrylic acid, and glycolic acid (Singh et al, 2006; Gong et al, 2012)
Models of cyanide dihydratase from Bacillus pumilus (CynD) based on the co-ordinates of either the nitrilaselike beta-alanine synthase from Drosophila melanogaster (PDB ID:2VHI), or the oxy-nitrilase from Synechocystis sp. strain PCC6803 (PDB ID:3WUY), were generated using the alignments shown in Figure 2 using Modeller (Sali and Blundell, 1993) running within UCSF Chimera (Pettersen et al, 2004)
The crystal structures of β-alanine synthase (βaS) (PDB id: 2VHI) and the oxynitrilase (PDB id: 3WUY) both show the twofold symmetry of the interactions across the C-surface, but in most respects the nature of the interactions and the conformation of the common insertion are different (Figure 3)

Summary

Introduction

Nitrilase enzymes from the large nitrilase-superfamily are of significant industrial interest due to their ability to process nitrile compounds into valuable acid products such as nicotinic acid, acrylic acid, and glycolic acid (Singh et al, 2006; Gong et al, 2012). They offer economic and environmentally friendly alternatives to current hazardous and costly methods of detoxifying nitrile wastes (Korte and Coulston, 1995; Gong et al, 2012).

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