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  • Characterization of the nickel-inserting cyclometallase LarC from Moorella thermoacetica and identification of a cytidinylylated reaction intermediate
Title: Characterization of the nickel-inserting cyclometallase LarC from Moorella thermoacetica and identification of a cytidinylylated reaction intermediate
Abstract LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.  more » « less
Award ID(s):
1807073
PAR ID:
10364454
Author(s) / Creator(s):
; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Metallomics
Volume:
14
Issue:
3
ISSN:
1756-591X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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