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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Biochem J. 2009 Sep 14;423(1):41–52. doi: 10.1042/BJ20090786

Figure 4. Reconstitution of Dictyostelium GDP-Fucose biosynthesis in vitro.

Figure 4

(A) Recombinant forms of the Dictyostelium GMD and GER encoded by cDNAs derived from either the AX3 or the HL250 strain were assayed in the following combinations: 1, AX3 GMD with AX3 GER; 2, HL250 GMD with AX3 GER; 3, AX3 GMD with HL250 GER; 4, HL250 GMD with HL250 GER; 5, overlaid chromatograms of GDP-Man and GDP-Fuc standards; other peaks in the RP-HPLC chromatograms derive from the bacterial lysates. (B) Expression of protein from all four clones was demonstrated by anti-His Western blotting: 1, AX3 GMD; 2, AX3 GER; 3, HL250 GMD; 4, HL250 GER. (C) Comparison of the protein sequences of various GDP-Man dehydratases in the region surrounding the site corresponding to the mutation in the HL250 GMD cDNA; DdA, Dicytostelium discoideum AX3 (residues 98-117); DdH, Dicytostelium discoideum HL250; Ce1, Caenorhabditis elegans GMD-1 (BRE-1); Dm, Drosophila melanogaster GMD; Hs, Homo sapiens GMD; Ec, Escherichia coli GMD; At1, Arabidopsis thaliana GMD1. Amino acids identical in a majority of the selected sequences are highlighted and the Gly → Asp change in the HL250 sequence is marked with an asterisk. On the other hand, the AX3 and HL250 ger cDNA sequences were found to be identical, although expression and activity levels differed in the experiments shown in panels A and B.