Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases.
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 Mg(2+) 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.
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland. martina.andberg@vtt.fi.
Martina
M
Andberg
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Niina
N
Aro-Kärkkäinen
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Paul
P
Carlson
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Merja
M
Oja
INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, F-31077, Toulouse, France.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400, Toulouse, France.|CNRS, UMR5504, F-31400, Toulouse, France.
Sophie
S
Bozonnet
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Mervi
M
Toivari
Department of Chemistry, University of Eastern Finland, PO Box 111, FI-80101, Joensuu, Finland.
Nina
N
Hakulinen
INSA, UPS, INP; LISBP, Université de Toulouse, 135 Avenue de Rangueil, F-31077, Toulouse, France.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400, Toulouse, France.|CNRS, UMR5504, F-31400, Toulouse, France.
Michael
M
O'Donohue
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Merja
M
Penttilä
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, VTT, FI-02044, Espoo, Finland.
Anu
A
Koivula
D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism*; D016961:Rhizobium leguminosarum / Q000201:enzymology*; D016415:Sequence Alignment; D014994:Xylose / Q000378:metabolism
D001426:Bacterial Proteins; D005942:Gluconates; D014994:Xylose; D001089:Arabinose; C037004:D-xylo-aldonate dehydratase; D006836:Hydro-Lyases; C030691:gluconic acid
D-xylonate dehydratase; EC 4.2.1.25; EC 4.2.1.82; IlvD/EDD family; L-arabonate dehydratase