Edited by Barry HalliwellKeywords: Phenylalanine hydroxylase Tetrahydrobiopterin Tetrahydrodictyopterin Dihydropteridine reductase Dihydrofolate reductase Dictyostelium a b s t r a c t Dictyostelium discoideum Ax2 produces both L-erythro-tetrahydrobiopterin (BH4) and its stereoisomer D-threo-BH4 (DH4). The putative cofactor function of them for phenylalanine hydroxylase (PAH) was investigated through genetic manipulation and quantitative determination of pteridines. In addition to establishing that dihydropteridine reductase (DHPR) and dihydrofolate reductase (DHFR) constitute the regeneration pathway of both BH4 and DH4, the results suggested that BH4 is a preferential cofactor for PAH in vivo, not a secondary product of DH4, which functions mainly as an antioxidant. Our result also demonstrated that PAH may be essential for Dictyostelium growth in nature, and thus it appears that the organism has evolved a strategy to maintain BH4 level via regeneration pathway at the expense of DH4 under oxidative stress conditions.

Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.

CY-007 and CY-049 pteridine glycosyltransferases (PGTs) that differ in sugar donor specificity to catalyze either glucose or xylose transfer to tetrahydrobiopterin were studied here to uncover the structural determinants necessary for the specificity. The importance of the C-terminal domain and its residues 218 and 258 that are different between the two PGTs was assessed via structure-guided domain swapping or single and dual amino acid substitutions. Catalytic activity and selectivity were altered in all the mutants (2 chimeric and 6 substitution) to accept both UDP-glucose and UDP-xylose. In addition, the wild type activities were improved 1.6-4.2 fold in 4 substitution mutants and activity was observed towards another substrate UDP-Nacetylglucosamine in all the substitution mutants from CY-007 PGT. The results strongly support essential role of the C-terminal domain and the two residues for catalysis as well as sugar donor specificity, bringing insight into the structural features of the PGTs. [BMB Reports 2013; 46(1): 37-40]