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...
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Veröffentlicht in: | Applied microbiology and biotechnology 2016-09, Vol.100 (17), p.7549-7563 |
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Sprache: | eng |
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Zusammenfassung: | 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 (k
cat
) but not for the substrate binding (K
m
) of the two pentonate dehydratases. |
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ISSN: | 0175-7598 1432-0614 |
DOI: | 10.1007/s00253-016-7530-8 |