TY  - JOUR
A1  - Gutt, Miriam
A1  - Jordan, Britta
A1  - Weidenbach, Katrin
A1  - Gudzuhn, Mirja
A1  - Kiessling, Claudia
A1  - Cassidy, Liam
A1  - Helbig, Andreas J.
A1  - Tholey, Andreas
A1  - Pyper, Dennis Joshua
A1  - Kubatova, Nina
A1  - Schwalbe, Harald
A1  - Schmitz-Streit, Ruth
T1  - High complexity of glutamine synthetase regulation in Methanosarcina mazei: small protein 26 interacts and enhances glutamine synthetase activity
T2  - The FEBS journal
N2  - Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 0.76 µm ± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1. Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.
KW  - glutamine synthetase regulation
KW  - Methanosarcina mazei
KW  - nitrogen regulation
KW  - small ORFs
KW  - small proteins
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/61821
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-618216
SN  - 1742-4658
IS  - Online Version of Record before inclusion in an issue
SP  - 1
EP  - 24
PB  - Wiley-Blackwell
CY  - Oxford
ER  -