The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification

Micah T. Nelp, Andrei V. Astashkin, Linda A. Breci, Reid M. McCarty, Vahe Bandarian

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.

Original languageEnglish (US)
Pages (from-to)3990-3994
Number of pages5
JournalBiochemistry
Volume53
Issue number24
DOIs
StatePublished - Jun 24 2014

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Toyocamycin
Post Translational Protein Processing
Catalyst activity
Streptomyces rimosus
Catalytic Domain
Nitriles
Niacin
Biosynthesis
Substrates
Substrate Specificity
Cobalt
nitrile hydratase
Kinetic parameters
Scaffolds
Hydration
Purification
Paramagnetic resonance
Iron
Anti-Bacterial Agents
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification. / Nelp, Micah T.; Astashkin, Andrei V.; Breci, Linda A.; McCarty, Reid M.; Bandarian, Vahe.

In: Biochemistry, Vol. 53, No. 24, 24.06.2014, p. 3990-3994.

Research output: Contribution to journalArticle

Nelp, Micah T. ; Astashkin, Andrei V. ; Breci, Linda A. ; McCarty, Reid M. ; Bandarian, Vahe. / The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification. In: Biochemistry. 2014 ; Vol. 53, No. 24. pp. 3990-3994.
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