Studies of the Redox Properties of CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1) and CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase reductase (E3): Two Important Enzymes Involved in the Biosynthesis of Ascarylose

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Abstract
Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5‘-phosphate dependent iron−sulfur protein. The second catalyst, CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-Δ3,4-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be −212 mV, with the FADox/FADsq couple (E1°‘) and the FADsq/FADhq couple (E2°‘) calculated to be −231 and −192 mV, respectively. However, the E1°‘ and E2°‘ of the FAD in E3(apoFeS) at pH 7.5 were estimated to be −215 and −240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron−sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron−sulfur is −257 mV and that of the E1 [2Fe-2S] center is −209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH → FAD → E3[2Fe-2S] → E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron−sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to −273 mV (E1°‘ ≈ −345 mV and E2°‘ ≈ −200 mV) at pH 8.4, with very little semiquinone stabilization (3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to −279 mV, and that of the E1 iron sulfur center remained essentially the same at −206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.