Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characteri...

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Autores principales:	Fiona F. Hager-Mair, Cordula Stefanović, Charlie Lim, Katharina Webhofer, Simon Krauter, Markus Blaukopf, Roland Ludwig, Paul Kosma, Christina Schäffer
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	cell wall glycopolymer enzyme assay kinetic constants pyruvyltransferase substrate synthesis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0c6bf112af424575b757ea469fbc5f0a
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spelling	oai:doaj.org-article:0c6bf112af424575b757ea469fbc5f0a2021-11-25T16:54:36ZAssaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release10.3390/biom111117322218-273Xhttps://doaj.org/article/0c6bf112af424575b757ea469fbc5f0a2021-11-01T00:00:00Zhttps://www.mdpi.com/2218-273X/11/11/1732https://doaj.org/toc/2218-273XKetalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from <i>Paenibacillus alvei</i>, a β-<span style="font-variant: small-caps;">d</span>-ManNAc-α-<span style="font-variant: small-caps;">d</span>-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a <i>K</i><sub>M, PEP</sub> of 19.50 ± 3.50 µM and <i>k</i><sub>cat, PEP</sub> of 0.21 ± 0.01 s<sup>−1</sup> as well as a <i>K</i><sub>M, Acceptor</sub> of 258 ± 38 µM and a <i>k</i><sub>cat, Acceptor</sub> of 0.15 ± 0.01 s<sup>−1</sup> were revealed. <i>P. alvei</i> CsaB was inactive on synthetic <i>p</i>NP-β-<span style="font-variant: small-caps;">d</span>-ManNAc and β-<span style="font-variant: small-caps;">d</span>-ManNAc-β-<span style="font-variant: small-caps;">d</span>-GlcNAc-1-<i>O</i>Me, supporting the necessity of a complex acceptor substrate.Fiona F. Hager-MairCordula StefanovićCharlie LimKatharina WebhoferSimon KrauterMarkus BlaukopfRoland LudwigPaul KosmaChristina SchäfferMDPI AGarticlecell wall glycopolymerenzyme assaykinetic constantspyruvyltransferasesubstrate synthesisMicrobiologyQR1-502ENBiomolecules, Vol 11, Iss 1732, p 1732 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell wall glycopolymer enzyme assay kinetic constants pyruvyltransferase substrate synthesis Microbiology QR1-502
spellingShingle	cell wall glycopolymer enzyme assay kinetic constants pyruvyltransferase substrate synthesis Microbiology QR1-502 Fiona F. Hager-Mair Cordula Stefanović Charlie Lim Katharina Webhofer Simon Krauter Markus Blaukopf Roland Ludwig Paul Kosma Christina Schäffer Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
description	Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from <i>Paenibacillus alvei</i>, a β-<span style="font-variant: small-caps;">d</span>-ManNAc-α-<span style="font-variant: small-caps;">d</span>-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a <i>K</i><sub>M, PEP</sub> of 19.50 ± 3.50 µM and <i>k</i><sub>cat, PEP</sub> of 0.21 ± 0.01 s<sup>−1</sup> as well as a <i>K</i><sub>M, Acceptor</sub> of 258 ± 38 µM and a <i>k</i><sub>cat, Acceptor</sub> of 0.15 ± 0.01 s<sup>−1</sup> were revealed. <i>P. alvei</i> CsaB was inactive on synthetic <i>p</i>NP-β-<span style="font-variant: small-caps;">d</span>-ManNAc and β-<span style="font-variant: small-caps;">d</span>-ManNAc-β-<span style="font-variant: small-caps;">d</span>-GlcNAc-1-<i>O</i>Me, supporting the necessity of a complex acceptor substrate.
format	article
author	Fiona F. Hager-Mair Cordula Stefanović Charlie Lim Katharina Webhofer Simon Krauter Markus Blaukopf Roland Ludwig Paul Kosma Christina Schäffer
author_facet	Fiona F. Hager-Mair Cordula Stefanović Charlie Lim Katharina Webhofer Simon Krauter Markus Blaukopf Roland Ludwig Paul Kosma Christina Schäffer
author_sort	Fiona F. Hager-Mair
title	Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
title_short	Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
title_full	Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
title_fullStr	Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
title_full_unstemmed	Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release
title_sort	assaying <i>paenibacillus alvei</i> csab-catalysed ketalpyruvyltransfer to saccharides by measurement of phosphate release
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/0c6bf112af424575b757ea469fbc5f0a
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Assaying <i>Paenibacillus alvei</i> CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

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