Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology submitted time 2016-05-12
Fan, Shanghua
Chen, Guanjun
Li, De-Feng
Wang, Da-Cheng
Fleming, Joy
Zhang, Hongtai
Zhou, Ying
Zhang, Xian-En
Bi, Lijun
Li, De-Feng
Wang, Da-Cheng
Fleming, Joy
Zhang, Hongtai
Zhou, Ying
Zhang, Xian-En
Bi, Lijun
Zhou, Lin
Chen, Tao
Zhou, Jie
Abstract： The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5'-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glul 71 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1-37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway. (C) 2014 Elsevier Ltd. All rights reserved.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics submitted time 2016-05-11
Zhou, Ying
Fleming, Joy
Wang, Liwei
Wang, Yingying
Zhang, Xiaoli
Wei, Wenjing
Bi, Lijun
Chen, Tao
Zhou, Lin
Deng, Jiaoyu
Wang, Xude
Abstract： Lysine acetylation is an important post-translational modification and is known to regulate many eukaryotic cellular processes. Little, however, is known about acetylated proteins in prokaryotes. Here, using immunoblotting, mass spectrometry and mutagenesis studies, we investigate the acetylation dynamics of the DNA repair protein Ku and its relationship with the deacetylase protein Sir2 and the non-homologous end joining (NHEJ) pathway in Mycobacterium smegmatis. We report that acetylation of Ku increases with growth, while NHEJ activity decreases, providing support for the hypothesis that acetylation of Ku may be involved in the DNA damage response in bacteria. Ku has multiple lysine sites. Our results indicate that K29 is an important acetylation site and that deficiency of Sir2 or mutation of K29 affects the quantity of Ku and its acetylation dynamics. Our findings expand knowledge of acetylation targets in prokaryotes and indicate a new direction for further research on bacterial DNA repair mechanisms.
Peer Review Status:Awaiting Review
Hits
Hits
Downloads
Comment