Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases

Research output: Contribution to journalArticle

Researchers

  • Martina Andberg
  • Niina Aro-Kärkkäinen
  • Paul Carlson
  • Merja Oja
  • Sophie Bozonnet
  • Mervi Toivari
  • Nina Hakulinen
  • Michael O'Donohue
  • Merja Penttilä

  • Anu Koivula

Research units

  • VTT Technical Research Centre of Finland
  • Institut national de la recherche agronomique
  • CNRS UMR 5504
  • Institut national des sciences appliquées de Toulouse
  • University of Eastern Finland

Abstract

We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg2+ for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (k(cat)) but not for the substrate binding (K-m) of the two pentonate dehydratases.

Details

Original languageEnglish
Pages (from-to)7549-7563
Number of pages15
JournalApplied Microbiology and Biotechnology
Volume100
Issue number17
Publication statusPublished - Sep 2016
MoE publication typeA1 Journal article-refereed

    Research areas

  • D-xylonate dehydratase, L-arabonate dehydratase, IlvD/ EDDfamily, EC 4.2.1.82, EC 4.2.1.25, DIHYDROXY-ACID DEHYDRATASE, D-MANNONATE DEHYDRATASE, L-ARABINOSE METABOLISM, D-XYLONATE PRODUCTION, D-XYLOSE METABOLISM, ENOLASE SUPERFAMILY, ENZYMATIC-ACTIVITIES, ESCHERICHIA-COLI, SACCHAROMYCES-CEREVISIAE, CAULOBACTER-CRESCENTUS

ID: 9224979