分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 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
摘要： 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.
分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-12
Zhang, Xiao-Li
Zhang, Xian-En
Zhang, Xiao-Li
Li, De-Feng
Fleming, Joy
Wang, Li-Wei
Zhou, Ying
Wang, Da-Cheng
Zhang, Xian-En
Bi, Li-Jun
Zhang, Xiao-Li
Li, De-Feng
Fleming, Joy
Wang, Li-Wei
Zhou, Ying
Wang, Da-Cheng
Zhang, Xian-En
Bi, Li-Jun
Zhang, Xiao-Li
摘要： Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-DN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-DN72 that assemble together between 2 membrane proximal domains of the EccB1-DN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.
分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-12
Fan Shang-Hua
Yu Zi-Niu
Fan Shang-Hua
Zhou Ying
Zhang Hong-Tai
Fleming, Joy
Zhang Xian-En
Bi Li-Jun
Fan Shang-Hua
Zhou Ying
Zhang Hong-Tai
Fleming, Joy
Zhang Xian-En
Bi Li-Jun
摘要： MutM (Formamidopyrimidine-DNA glycosylase, Fpg), a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase), is involved in the repair of many kinds of DNA damage, including the formation of 8-oxoguanine, 5-formyluracil, and C/C mismatches, through recognizing DNA damage and removing damaged bases. The mechanisms of MutM involvement, however, with the exception of 8-oxoG, are poorly understood. Here, we identified proteins which interact with MutM in Mycobacterium smegmatis using methods of tandem affinity purification and mass spectrometry and used Far-western and GST pull-down analysis to validate the interactions between MutM and DEAD-box rna helicase, RpsC, and UvrA. Results demonstrated that tandem affinity purification is a suitable method for identifying MutM interacting proteins and provided insights into the mechanism by which MutM is involved in DNA damage repair.
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