אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

תקצירי הכינוס השנתי ה-47 | 2013

ישיבה שביעית: שחפת, מחלה המתעוררת מחדש

פיתוח תרופות חדשות כנגד שחפת

ד"ר אורן צמחוני,
היחידה למחלות זיהומיות, המרכז הרפואי קפלן, רחובות

Tuberculous phospho-pantothenic transferase PptT activates AcpM, the acyl carrier protein for mycolic acid biosynthesis in mycobacteria.

Oren Zimhony1* Alon Shwartz2*, Yoav peleg3, Orly Dym3, Shira Albeck3  Yigal Burstein4 and Zippi Shakked2
1. Kaplan Medical Center, affiliated to the school of Medicine Hebrew University Jerusalem POB 1 Rehovot Israel ,
2. Structral Biology Department, 3Israeli Structural Proteomic Center, 4Organic Chemistery Department,
 Weizmann Institute of Science, Rehovot, Israel.

Drug resistant tuberculosis is widely distributed and is a major threat.  Fatty acid biosynthesis is an essential process for cell survival, and is particularly proficient and complex for mycobacteria and related species that produce mycolic acids, the main component of mycobacterial cell wall, which plays a major role in virulence and drug resistance.  Various steps in the synthesis of mycolates are targets for first and second-line anti-tuberculosis agents. Mycobacterial fatty acid synthase 1(FAS-I) generates and elongates long chain (C16:0-26:0) acyl-CoA derivatives that serve as the precursors for the syntheses of polyketides and C56:0 fatty acids designated as meromycolate by fatty acid synthase 2 (FAS-II). The elongated acyl intermediates throughout fatty acid synthesis are attached to an acyl carrier protein (ACP).

The acyl intermediates in fatty acid and mycolic acid synthesis are bound to an acyl carrier protein (ACP) domain within the multifunctional fatty acid synthase I (FAS I) and polyketide synthases (PKS), or to an ACP protein of the FAS-II system (ACPM). The inactive apo-form of ACPs is converted to the holo-form by the covalent attachment of a 4’-phosphopantetheinyl moiety of CoA to a conserved serine residue mediated by a phosphopantetheine transferase (PPTase).

Two genes encoding PPTases, Rv2523 and Rv2794,  were identified in M. tuberculosis H37Rv encoding: AcpS which activates FAS I, and PptT which activates several PKSs, ACPM was assumed to be activated by AcpS, as in other bacterial species. Both PPTases were found to be essential. Analysis of the AcpS structure, revealed that unlike other AcpS-es it is negatively charged and lacks key basic residues that were reported to allow interaction with ACP. We subjected purified apo-ACPM to AcpS or to PptT in-vitro, and assesed their bioactivity by the band shift of holo-ACPM using SDS-PAGE followed by mass spectrometry. Interestingly, we found that PptT activates AcpM, like B.Subtilis PPTase SfpNEB while AcpS did not activate ACPM. Phosphopantetheine transfer to ACPM by PptT showed typical kinetics, was CoA dependent and ACPM S41A was not activated. Thus, PptT is likely the principle PPTase activator for meromycolic acid synthesis, condensation and modification. PptT activates a blue pigment synthase BPSA which is a non ribosomal peptide synthase(NRPS) from  Streptomyces lavandulae.  Activated BpsA produces indigoidine a blue pigment that enable rapid and flexible assessment of PptT activity and screen for new inhibitors of PptT that can be  used as novel therapeutics for  resistant Mtb.  


*Equal contribution

חזרה לתקצירי הכנס
דף הבית | מי אנחנו | רישום לאילמ"ר ולאתר | תקנון | חברי הועד | מערכת האתר | צור קשר | מקצוע המעבדנות הרפואית | מישיבות ועד אילמ"ר | המועצה למעבדות במשה"ב |
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