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Journal articles
Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center
Abstract : PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.
Keywords :
8-anilino-1-naphthalenesulfonic acid ammonium salt
9- and 10-nitro-9-cis-octadecaenoic acids
9-octadecenoic acid
ANS
BPS
ESI
IAM
IPTG
LC
Mycobacterium tuberculosis
Nitrated fatty acids
Nitroalkene
OA oleic acid
OA-NO(2) nitro-oleic acid
PknG
Rbx
Rubredoxin
Ser/Thr kinase
bathophenanthroline disulfonate
electrospray ionization
iodoacetamide
isopropyl β-D-1-thiogalactopyranoside
liquid chromatography
rubredoxin
https://hal-pasteur.archives-ouvertes.fr/pasteur-03137890
Contributor : Marie de Tarragon Connect in order to contact the contributor
Submitted on : Wednesday, February 10, 2021 - 5:13:49 PM
Last modification on : Tuesday, January 18, 2022 - 2:50:08 PM
Contributor : Marie de Tarragon Connect in order to contact the contributor
Submitted on : Wednesday, February 10, 2021 - 5:13:49 PM
Last modification on : Tuesday, January 18, 2022 - 2:50:08 PM
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