9d5u
Nitrile hydratase S112A mutant
Structural highlights
FunctionNHAA_PSETH NHase catalyzes the hydration of various nitrile compounds to the corresponding amides. Publication Abstract from PubMedA highly conserved second-sphere active site alphaSer residue in nitrile hydratase (NHase), that forms a hydrogen bond with the axial metal-bound water molecule, was mutated to Ala, Asp, and Thr, in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Thr in the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). All five mutants were successfully purified; metal analysis via ICP-AES indicated that all three Co-type PtNHase mutants were in their apo-form while the Fe-type alphaSer117Ala and alphaSer117Thr mutants contained 85 and 50 % of their active site Fe(III) ions, respectively. The k(cat) values obtained for the PtNHase mutant enzymes were between 0.03 +/- 0.01 and 0.2 +/- 0.02 s(-1) amounting to <0.8 % of the k(cat) value observed for WT PtNHase. The Fe-type ReNHase mutants retained some detectable activity with k(cat) values of 93 +/- 3 and 40 +/- 2 s(-1) for the alphaSer117Ala and alphaSer117Thr mutants, respectively, which is approximately 5 % of WT ReNHase activity towards acrylonitrile. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzymes showed subtle changes in the electronic environment around the active site Fe(III) ions, consistent with altering the hydrogen bonding interaction with the axial water ligand. X-ray crystal structures of the three PtNHase mutant enzymes confirmed the mutation and the lack of active site metal, while also providing insight into the active site hydrogen bonding network. Taken together, these data confirm that the conserved active site alphaSer residue plays an important catalytic role but is not essential for catalysis. They also confirm the necessity of the conserved second-sphere alphaSer residue for the metalation process and subsequent post-translational modification of the alpha-subunit in Co-type NHases but not Fe-type NHases, suggesting different mechanisms for the two types of NHases. SYNOPSIS: A strictly conserved active site alphaSer residue in both Co- and Fe-type nitrile hydratases was mutated. This alphaSer residue was found to play an important catalytic function, but is not essential. In Co-type NHases, it appears to be essential for active site maturation, but not in Fe-type NHases. Catalytic and post-translational maturation roles of a conserved active site serine residue in nitrile hydratases.,Miller C, Knutson K, Liu D, Bennett B, Holz RC J Inorg Biochem. 2024 Oct 18;262:112763. doi: 10.1016/j.jinorgbio.2024.112763. PMID:39447484[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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