The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.

2.50
HDL Handle:
http://hdl.handle.net/10033/92920
Title:
The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.
Authors:
Elamin, Ayssar A; Stehr, Matthias; Oehlmann, Wulf; Singh, Mahavir
Abstract:
The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K(m) and K(cat) were determined with values of 129.6+/-8.1 microM and 65.4+/-4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.
Affiliation:
Department of Genome Analysis, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Citation:
The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall. 2009, 79 (3):358-63 J. Microbiol. Methods
Journal:
Journal of microbiological methods
Issue Date:
Dec-2009
URI:
http://hdl.handle.net/10033/92920
DOI:
10.1016/j.mimet.2009.10.010
PubMed ID:
19857528
Type:
Article
Language:
en
ISSN:
1872-8359
Appears in Collections:
Publications of Dept. Genome Analysis (GNA)

Full metadata record

DC FieldValueLanguage
dc.contributor.authorElamin, Ayssar Aen
dc.contributor.authorStehr, Matthiasen
dc.contributor.authorOehlmann, Wulfen
dc.contributor.authorSingh, Mahaviren
dc.date.accessioned2010-02-24T13:30:33Z-
dc.date.available2010-02-24T13:30:33Z-
dc.date.issued2009-12-
dc.identifier.citationThe mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall. 2009, 79 (3):358-63 J. Microbiol. Methodsen
dc.identifier.issn1872-8359-
dc.identifier.pmid19857528-
dc.identifier.doi10.1016/j.mimet.2009.10.010-
dc.identifier.urihttp://hdl.handle.net/10033/92920-
dc.description.abstractThe enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K(m) and K(cat) were determined with values of 129.6+/-8.1 microM and 65.4+/-4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.en
dc.language.isoenen
dc.titleThe mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.en
dc.typeArticleen
dc.contributor.departmentDepartment of Genome Analysis, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.en
dc.identifier.journalJournal of microbiological methodsen

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