Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases

Abstract

A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase

“…Although the FLS2 gene is transcribed, it is fairly unlikely that the resulting protein has FLS activity. The deduced polypeptide from the FLS2 transcript is missing the C-terminal protein part (Supplementary Figure 1), including the last histidine residue of the HxD…H motiv involved in 2-oxoglutarate binding (Britsch et al 1993;Roach et al 1995;Lukacin and Britsch 1997) as well as the amino acid residues involved in iron binding (Myllylä et al 1992;Britsch et al 1993;Lukacin and Britsch 1997;Clifton et al 2006). Taken together, these results indicate that only FLS1, FLS3 and FLS5 encode full-length proteins.…”

Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

“…In addition, the "His-2 motif" (amino acids 679-697), present in ␣-ketoglutarate-dependent dioxygenases (8,24) and containing a histidine essential for Fe 2ϩ /␣-ketoglutarate binding (8), is identical. Finally, in a fluorescence in situ hybridization analysis for subchromosomal localization of the gene, all 80 of the metaphase cells examined by in situ hybridization exhibited specific labeling of the proximal long arm of chromosome 8.…”

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

“…Higher amino acid-sequence homology might be expected between the different GA,2-oxoglutarate dioxygenases, although at present no other sequences are available for comparison. The derived sequence contains three conserved histidines, which may serve to bind Fe at the enzyme active center (17,20,21). The sequence contains no recognizable N-terminal targeting signals for particular cell compartments.…”