Bacterial nitrilases and their regulation.

Commercially, nitrilases are valuable biocatalysts capable of converting a diverse range of nitriles to carboxylic acids for the greener synthesis of chemicals and pharmaceuticals. Nitrilases are widespread in nature and are both important components of metabolic pathways and a response to environmental factors such as natural or manmade nitriles. Nitrilases are often grouped together on a genome in specific gene clusters that reflect these metabolic functions. Although nitrilase induction systems are still poorly understood, it is known that a powerful Rhodococcal transcription regulator system permits accumulation of intracellular nitrilase of up to 30-40% of total soluble protein in wild type Rhodococcous rhodochrous and host Streptomyces strains. Nitrilase expression inducer molecules encompass a broad range of aliphatic, aromatic and heteroaromatic nitriles, as well as some secondary and tertiary amides that are resistant to nitrilase degradation.

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer.

BACKGROUND: The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. RESULTS: A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. CONCLUSIONS: The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.