To investigate the molecular basis of specificity in B. pallidus nitrile hydratase by means of X-ray crystallography.
The nitrile hydratase (NHase) enzyme family, catalyzing the conversion of organocyanides to their corresponding amides, is widespread in mesophilic representatives of the bacterial and eukaryotic kingdoms. However, only five thermophilic NHase producing organisms have thus far been described, four of which belong to the genus Bacillus () () (), while the fifth is designated as Pseudonocardia thermophila.
Despite significant differences in origin, stability, cofactor requirements and catalytic characteristics between the various NHase sub-groups, there is a high degree of similarity in terms of size and protein sequence. It is believed therefore that all enzymes in the NHase family have very similar structures and catalytic mechanisms (). Typically, NHases are heteromultimers composed of two distinct subunits, designated a and b. The crystal structures of two mesophilic NHases (photoactivated Rhodococcus sp. R312 NHase () and nitrosylated NHase from Rhodococcus sp. N-771 () have been obtained at 2.3 and 1.7, respectively. A native structure has been determined from another thermophile, Pseudonorcardia thermophila. This nitrile hydratase has 59% (alpha subunit) and 40% (beta subunit) amino acid identity (77% and 64% aa similarity respectively) with our Bacillus NHase.
The enzymatic conversion of acrylonitrile to acrylamide is one of the more successful applications of biotechnology in commodity chemical production and has unarguably demonstrated the commercial viability of NHase () (). However, the capacity of NHase enzyme systems to convert a cyano- functionality to an amide (or to an acid in the presence of an amidase) is also potentially valuable in the synthesis of numerous commodity and speciality chemicals. NHases showing novel stereoselectivity, regiospecificity or substrate specificity may be developed to produce amides that are components of pharmaceutical compounds ().
Information gained from structural analysis and understanding the molecular basis of the specificity of these enzymes would enable us to manipulate the enzyme rationally for use in industrial biotransformations.
The approach would be to firstly produce highly purified material of the native protein and various mutants for crystallization purposes. Exhaustive crystallization trials would be carried out with the aim of obtaining diffraction quality crystals. Initial X-ray diffraction test and data collection will be performed using the newly purchased in-house X-ray source part of SASBI. High resolution data will also be collected at synchrotron radiation facilities based overseas. After merging and processing data, model phases will be obtained using the Molecular Replacement method. The Rhodococcus nitrile hydratase crystal structure will be used as a probe during the molecular replacement search.