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The structure of the nitrile hydratase from Bacillus pallidus

Aims

To investigate the molecular basis of specificity in B. pallidus nitrile hydratase by means of X-ray crystallography.

Background

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  [cite num=1] [cite num=2] [cite num=3], 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 [cite num=4]. 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 [cite num=”5″] and nitrosylated NHase from Rhodococcus sp. N-771 [cite num=”6″] 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 [cite num=”7″] [cite num=”8″]. 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 [cite num=”9″].

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.

Methodology

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.

References

  1. Cramp, R. A., & Cowan, D. A. (1999). Molecular characterisation of a novel thermophilic nitrile hydratase. Biochim Biophys Acta, 1431(1), 249-60. PMID: 10209297 [cite num=”1″ return=TRUE]
  2. Kim, S., & Oriel, P. (2000). Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli. Enzyme Microb Technol, 27(7), 492-501. PMID: 10978771 [cite num=2 return=true]
  3. Pereira, R. A., Graham, D., Rainey, F. A., & Cowan, D. A. (1998). A novel thermostable nitrile hydratase. Extremophiles, 2(3), 347-57. PMID: 9783183 [cite num=3 return=true]
  4. Mascharak, P. K. (2002). Structural and functional models of nitrile hydratase.  Coord Chem Rev, 225, 201-14 [cite num=4 return=true]
  5. Huang, W., Jia, J., Cummings, J., Nelson, M., Schneider, G., & Lindqvist, Y. (1997). Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold. Structure, 5(5), 691-9. PMID: 9195885 [cite num=”5″ return=TRUE]
  6. Nagashima, S., Nakasako, M., Dohmae, N., Tsujimura, M., Takio, K., Odaka, M., . . . Endo, I. (1998). Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms. Nat Struct Biol, 5(5), 347-51. PMID: 9586994 [cite num=”6″ return=TRUE]
  7. Cowan, D., Cramp, R., Pereira, R., Graham, D., & Almatawah, Q. (1998). Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes. Extremophiles, 2(3), 207-16. PMID: 9783167 [cite num=”7″ return=TRUE]
  8. Kobayashi, M., Nagasawa, T., & Yamada, H. (1992). Enzymatic synthesis of acrylamide: a success story not yet over. Trends Biotechnol, 10(11), 402-8. PMID: 1368882 [cite num=”8″ return=TRUE]
  9. Kobayashi, M., & Shimizu, S. (1998). Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology. Nat Biotechnol, 16(8), 733-6. PMID: 9702770 [cite num=”9″ return=TRUE]