Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase

Area of Science:

• Biochemistry
• Enzymology
• Microbial Biotechnology

Background:

• Nitrilases are crucial enzymes catalyzing the hydrolysis of nitriles to carboxylic acids and ammonia.
• Understanding nitrilase mechanisms is vital for biocatalysis and industrial applications.
• Rhodococcus species are known producers of various enzymes, including nitrilases.

Purpose of the Study:

• To characterize the nitrilase produced by Rhodococcus ATCC 39484.
• To elucidate the catalytic mechanism and substrate specificity of this nitrilase.
• To investigate the enzyme's kinetic properties and inhibitor interactions.

Main Methods:

• Enzyme purification and characterization (subunit Mr, optimal pH, temperature stability).
• Substrate specificity profiling and kinetic analysis.
• Inhibitor studies (competitive and irreversible).
• Mass spectrometry to detect covalent enzyme-substrate intermediates.
• N-terminal sequencing for homology analysis.

Main Results:

• Purified nitrilase (40 kDa subunit) forms an activated 560 kDa complex.
• Broad substrate specificity with optimal activity at pH 7.5 and 40°C.
• N-terminal sequence shows homology to Klebsiella ozaenae nitrilase.
• Covalent intermediates (thioimidate or acylenzyme) detected via mass spectrometry.
• Reaction mechanism proposed, with intermediate breakdown being rate-limiting.
• Anomalous amide formation observed for phenylacetonitrile, suggesting a tetrahedral intermediate.

Conclusions:

• Rhodococcus ATCC 39484 nitrilase is a metalloenzyme with a complex activation mechanism.
• The enzyme's mechanism involves covalent intermediates and a potential tetrahedral intermediate.
• Characterization provides insights into nitrilase catalysis and potential for biocatalytic applications.