| Summary: | 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.
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