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 language | English |
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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 |
Keywords
- D-xylonate dehydratase
- EC 4.2.1.25
- EC 4.2.1.82
- IlvD/EDD family
- L-arabonate dehydratase