Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 7549-7563 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 100 |
| Issue number | 17 |
| DOIs | |
| Publication status | Published - 2016 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- D-xylonate dehydratase
- EC 4.2.1.25
- EC 4.2.1.82
- IlvD/EDD family
- L-arabonate dehydratase
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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 journal › Article › Scientific › peer-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 -