Characterization of a Distinct Arabinofuranosyltransferase in Mycobacterium smegmatis
详细信息 查看全文
- 作者：Jian Zhang ; Kay-Hooi Khoo ; Sz-Wei Wu ; Delphi Chatterjee
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：August 8, 2007
- 年：2007
- 卷：129
- 期：31
- 页码：9650 - 9662
- 全文大小：793K
- 年卷期：v.129,no.31(August 8, 2007)
- ISSN：1520-5126
文摘
The D-arabinans in Mycobacterium are essential, extraordinarily complex entity comprised ofD-arabinofuranose residues which are rarely found in nature. Despite the well-recognized importance ofthe mycobacterial arabinan, delineation of the arabinosylation process has been severely hampered dueto lack of positively identified arabinosyltransferases. Identification of genes involved in arabinan biosynthesisentailed the use of ethambutol (EMB), a first-line antituberculosis agent that is known to inhibit cell wallarabinan synthesis. The three genes (embA, embB, and embC) encode novel membrane proteins, implicatedas the only known mycobacterial arabinosyltransferases to this date. We have now adapted a multifacetedapproach involving development of convenient arabinosyltransferase assay using novel synthetic acceptorsto identify arabinosyltransferase/s that will be distinct from the Emb proteins. In our present work,Mycobacterium smegmatis mc2155 (WTMsm) was used as a model to study the biosynthesis of cell wallarabinan. In an in vitro assay, we demonstrate that transfer of only 
-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [-D-Araf]2-3,5--D-Araf-(1
5)--D-Araf-(15)--D-Araf (1) with an octyl aglycon. Higher molecular weight (up to Ara10) oligomerswere also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assistedlaser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of theseproducts revealed that isomeric products were formed and initiation and elongation of arabinan can occureither on the 5-arm or 3-arm of the branched 3,5--D-Araf. Individual embA, embB, and embC knockoutstrains retained this -1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol.This particular enzyme function is distinct from the function of the Emb proteins.
-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [-D-Araf]2-3,5--D-Araf-(15)--D-Araf-(15)--D-Araf (1) with an octyl aglycon. Higher molecular weight (up to Ara10) oligomerswere also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assistedlaser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of theseproducts revealed that isomeric products were formed and initiation and elongation of arabinan can occureither on the 5-arm or 3-arm of the branched 3,5--D-Araf. Individual embA, embB, and embC knockoutstrains retained this -1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol.This particular enzyme function is distinct from the function of the Emb proteins.