Mechanistic studies of nitrile hydratases and their activator proteins

Nitrile hydratases (NHases) are iron- or cobalt-containing metalloenzymes with a hexadentate active site characterized by a highly conserved CXXCSC motif on the α subunit. Two of the active site cysteine residues undergo post-translational oxidation to sulfenic and sulfinic acid moieties. Both the Fe-type and Co-type NHase require a metallochaperone or ‘activator’ protein for maturation to the fully functional state. Through spectroscopy, crystallography, and computational methods, this research elucidates key mechanistic features and provides novel insights into the NHase post-translation maturation process. The existing literature on NHase catalysis is rich with a significant amount of data establishing the first-coordination sphere. However, questions remain regarding second and third-sphere residues, proton transfer mechanisms, and substrate channels within NHases. This dissertation addresses these gaps by studying two strictly conserved second-sphere Arg residues on the β subunit, which proved to be highly important in NHase metalation and maturation. UV-Vis and EPR spectra indicate these residues are crucial for maintaining the correct Lewis acidity and catalytic turnover to occur. DFT calculations further proposed that these Arg residues stabilize the anionic nucleophile through hydrogen bonding. This work also investigates a conserved second-sphere Ser residue on the α subunit active site motif (CXXCSC), revealing its differentiating role in the Co-type and Fe-type NHases. Mutational studies of this residue provide the first evidence of the active site Ser's involvement in metalation and maturation, as well as its impact on the catalytic mechanism. Further, this dissertation presents the first characterization of a Co-type activator (ɛ) protein from Pseudonocardia thermophila JCM 3095. Using biochemical, bioinformatic, and spectroscopic techniques, this research proposes a high-spin Co(II) pentacoordinate metal center. This study also confirms the ATP/GTPase activity of the activator, aligning with previous findings. Collectively, this dissertation advances the understanding of NHase catalysis, emphasizing the important roles of second-sphere residues and presenting new information on the Co-type ɛ protein. The findings contribute to a more comprehensive model of NHase catalysis and maturation process and lay the groundwork for future studies on the enzyme's intricate mechanisms and potential industrial applications.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor dc:publisher

Colorado School of Mines. Arthur Lakes Library

Year dc:date.issued

2024

Author dc:creator

• Miller, Callie Caroline Grace

Advisor dc:contributor.advisor

• Holz, Richard C.

Committee members dc:contributor.committeemember

• Williams, S. Kim R.
• Domaille, Dylan
• Krebs, Melissa D.

• cobalt
• crystallography
• metallochaperone
• metalloenzyme
• nitrile hydratases
• spectroscopy

Statement dc:rights

• Copyright of the original work is retained by the author.

Language dc:language.iso

eng, English

Dc Identifier Other

T 9866

OAI identifier oai:identifier

oai:repository.mines.edu:11124/180297