2010
DOI: 10.1111/j.1742-4658.2010.07817.x
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Optimization of conditions for the glycosyltransferase activity of penicillin‐binding protein 1a from Thermotoga maritima

Abstract: Cell wall biosynthesis is a key target for antibacterial drugs. The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pressure. It consists of glycan chains of repeating disaccharide units crosslinked through short peptide chains. Peptidoglycan assembly is catalyzed by the periplasmic domain of bifunctional class A penicillin-binding proteins. Cross-linking of the peptide chains is catalyzed by their transpeptid… Show more

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Cited by 21 publications
(28 citation statements)
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“…the ⑀-amino group at the position 3 of the stem peptide is covalently modified with a dansyl group) (18). In this assay, polymerization of dansyl-lipid II followed by polymer digestion with a muramidase results in a decrease in fluorescence of the dansyl-muropeptides caused by the removal of the lipid moiety during polymerization (19). Although robust, this assay requires fluorescent derivitization of the limiting lipid II substrate (which is a challenge to attain in large quantities) and a secondary enzyme.…”
Section: Resultsmentioning
confidence: 99%
“…the ⑀-amino group at the position 3 of the stem peptide is covalently modified with a dansyl group) (18). In this assay, polymerization of dansyl-lipid II followed by polymer digestion with a muramidase results in a decrease in fluorescence of the dansyl-muropeptides caused by the removal of the lipid moiety during polymerization (19). Although robust, this assay requires fluorescent derivitization of the limiting lipid II substrate (which is a challenge to attain in large quantities) and a secondary enzyme.…”
Section: Resultsmentioning
confidence: 99%
“…Continuous fluorescence assay. TGase activity assays were performed as described (Schwartz et al ., 2002; Offant et al ., 2010) in a medium‐binding black 96‐well microplate (Greiner Bio One ref. 655076, Frickenhausen, Germany) using a FLUOstar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany).…”
mentioning
confidence: 99%
“…PBP1A (0.85 µM) was incubated with 4 µM radioactive [ 14 C]‐lipid II (0.126 µCi nmol −1 ) in the presence or absence of 4.5 µM PBP2 at 30°C for 3 or 20 min. The reaction mixtures (20 µl) contained 60 mM HEPES pH 7.5, 0.62 M NaCl, 0.1% Triton X‐100, 10 mM MgCl 2 , 20% DMSO and 1000 units ml −1 Penicillin G. Control samples contained no enzyme, 0.1 µM E. coli PBP1B (Terrak et al ., 1999) or 0.1 µM Thermotoga maritima PBP1a (Offant et al ., 2010). The reactions were stopped by the addition of moenomycin (10 µM) and analysed by 9% SDS‐PAGE (20 × 20 cm gels) as described (Barrett et al ., 2007).…”
mentioning
confidence: 99%
“…The presence of the dansyl group in the third position of the lipid II pentapeptide, prevented subsequent transpeptidation by bifunctional enzymes, allowing measurement of transglycosylation alone. This assay [41] has been converted to a multi-well format, which enables the rapid parallel screening of a range of reaction conditions [44]. This can allow, therefore, the screening and determination of optimal conditions for multiple transglycosylases from a range of microorganisms, essential in the study of these membrane proteins.…”
Section: Assays For Transglycosylase Activitymentioning
confidence: 99%