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

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

Research output: Contribution to journalArticleScientificpeer-review

14 Citations (Scopus)

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 (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.
Original languageEnglish
Pages (from-to)7549-7563
JournalApplied Microbiology and Biotechnology
Volume100
Issue number17
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

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Hydro-Lyases
Sulfur
Mutagenesis
Iron
Sugar Acids
Enzymes
Deoxy Sugars
Caulobacter crescentus
Rhizobium leguminosarum
Pentoses
Biomass
Cysteine
Amino Acid Sequence
Proteins
Escherichia coli
Acids

Keywords

  • D-xylonate dehydratase
  • EC 4.2.1.25
  • EC 4.2.1.82
  • IlvD/EDD family
  • L-arabonate dehydratase

Cite this

Andberg, Martina ; Aro-Kärkkäinen, Niina ; Carlson, Paul ; Oja, Merja ; Bozonnet, Sophie ; Toivari, Mervi ; Hakulinen, Niina ; O'Donohue, Michael ; Penttilä, Merja ; Koivula, Anu. / Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases. In: Applied Microbiology and Biotechnology. 2016 ; Vol. 100, No. 17. pp. 7549-7563.
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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 (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.",
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Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases. / Andberg, Martina; Aro-Kärkkäinen, Niina; Carlson, Paul; Oja, Merja; Bozonnet, Sophie; Toivari, Mervi; Hakulinen, Niina; O'Donohue, Michael; Penttilä, Merja; Koivula, Anu.

In: Applied Microbiology and Biotechnology, Vol. 100, No. 17, 2016, p. 7549-7563.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases

AU - Andberg, Martina

AU - Aro-Kärkkäinen, Niina

AU - Carlson, Paul

AU - Oja, Merja

AU - Bozonnet, Sophie

AU - Toivari, Mervi

AU - Hakulinen, Niina

AU - O'Donohue, Michael

AU - Penttilä, Merja

AU - Koivula, Anu

PY - 2016

Y1 - 2016

N2 - 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 (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.

AB - 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 (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.

KW - D-xylonate dehydratase

KW - EC 4.2.1.25

KW - EC 4.2.1.82

KW - IlvD/EDD family

KW - L-arabonate dehydratase

U2 - 10.1007/s00253-016-7530-8

DO - 10.1007/s00253-016-7530-8

M3 - Article

VL - 100

SP - 7549

EP - 7563

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 17

ER -