9d6k | pdb_00009d6k
Nitrile hydratase BR157A mutant
Structural highlights
FunctionNHAA_PSETH NHase catalyzes the hydration of various nitrile compounds to the corresponding amides. Publication Abstract from PubMedTwo conserved second-sphere betaArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase betaR52A, betaR52K, betaR157A, betaR157K and ReNHase betaR61A) were successfully expressed and purified. Apart from the PtNHase betaR52A mutant that exhibited no detectable activity, the k(cat) values obtained for the PtNHase and ReNHase betaR mutant enzymes were between 1.8 and 12.4 s(-1) amounting to <1% of the k(cat) values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from approximately 10 to approximately 40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase betaR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere betaR residues in the proposed subunit swapping process and post-translational modification of the alpha-subunit in the alpha activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form. Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.,Miller C, Huntoon D, Kaley N, Ogutu I, Fiedler AT, Bennett B, Liu D, Holz R J Inorg Biochem. 2024 Jul;256:112565. doi: 10.1016/j.jinorgbio.2024.112565. Epub , 2024 Apr 16. PMID:38677005[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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