Biosynthesis of a 3,6-dideoxyhexose: crystallization and X-ray diffraction of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) for ascarylose biosynthesis

Abstract

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), along with its reductase (E3), catalyzes the unusual C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose to form CDP-3,6-dideoxy-L-threo-D-glycero-4-hexulose in CDP-ascarylose biosynthesis [Chen et al. (1996), Biochemistry, 35, 16412-16420]. This dimeric [2Fe-2S] protein, cloned from the bacteria Yersinia pseudotuberculosis, is currently the only known example of an enzyme that uses a vitamin B6-derived pyridoxamine 5'-phosphate (PMP) cofactor to carry out one-electron chemistry [Agnihotri & Liu (2001), Bioorg. Chem. 29, 234-257]. It also exhibits a [2Fe-2S] cluster-binding motif (C-X57-C-X1-C-X7-C) which has not been observed previously [Agnihotri et al. (2004), Biochemistry, 43, 14265-14274] The recombinant 97.7 kDa dimer was crystallized in the trigonal space group P3(2), with unit-cell parameters a = b = 97.37, c = 142.2 A, alpha = beta = 90, gamma = 120 degrees. A data set has been collected to 1.9 A resolution. A full MAD data set was also collected at the iron absorption edge that diffracted to 2.0 A.

Figure 1

Deoxygenation of CDP-6-deoxy-l-threo-d-glycero-4-hexulose (1) to form CDP-3,6-dideoxy-l-threo-d-glycero-4-hexulose (2) catalyzed by E₁ dehydrase and E₃ reductase in CDP-ascarylose biosynthesis. Chemical steps within the E₁ active site are enclosed by a blue border and those undergone in the redox partner E₃ are enclosed by a pink border. Substrate (1) forms a Schiff base in the E₁ active site with the PMP cofactor, interconverting between the hydrated and dehydrated substrate adduct. The product is released upon two single-electron transfers from E₃. NADH is the original electron donor.

Figure 2

Hexagonal crystals of E₁, grown at 295 K for one week, are approximately 0.1 mm in length.