Identification of an unusual [2Fe-2S]-binding motif in the CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase from Yersinia pseudoutberculosis:: Implication for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E-1) catalyzes the C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. E-1 is a pyridoxamine 5'-phosphate (PMP)-dependent enzyme that also contains a [2Fe-2S] center. This iron-sulfur cluster is catalytically essential, since removal of the [2Fe-2S] center leads to inactive enzyme. To identify the [2Fe-2S] core in E-1 and to study the effect of impairing the iron-sulfur cluster on the activity of E-1, a series of E-1 cysteine mutants were constructed and their catalytic properties were characterized. Our results show that E-1 displays a cluster-binding Motif (C-X-57-C-X-1-C-X-7-C) that has not been observed previously for [2Fe-2S] proteins. The presence of such an unusual iron-sulfur cluster in E-1, along with the replacement of the active site lysine by a histidine residue (H220), reflects a distinct evolutionary path for this enzyme. The cysteine residues (C193, C251, C253, C261) implicated in the binding of the iron-sulfur cluster in E-1 are conserved in the sequences of its homologues. It is likely that E-1 and its homologues constitute a new subclass in the family of iron-sulfur proteins, which are distinguished not only by their cluster ligation patterns but also by the chemistry used in catalyzing a simple, albeit mechanistically challenging, reaction.