Wild-type and mutant 2,2-dialkylglycine decarboxylases: The catalytic role of active site glutamine 52 investigated by site-directed mutagenesis and computer analysis

Abstract

The ability of pyridoxal 5$\sp\prime$-phosphate (PLP)-dependent 2,2-dialkylglycine decarboxylase (DGD) to catalyze decarboxylation and transamination of amino acids at a single active site depends on the subsite within the active site that cleaves $\alpha$-H and $\alpha$-COO$\sp-$ bonds. As observed in the crystal structure, the strategic position of glutamine 52 at the active site suggests a role in enhancing decarboxylation via formation of a hydrogen bond to the substrate carboxyl group. Supporting evidence for this hypothesis is provided by studies with glutamine 52 active site mutants, computer modeling and protein sequence analyses. Ten mutant DGDs containing alanine, asparagine, aspartate, arginine, glutamate, glycine, histidine, leucine, lysine, and tryptophan at position 52 were produced. All, except the histidine mutant, exhibited decreased rates of decarboxylation compared to wild-type. Histidine and asparagine mutants showed measurable decarboxylation rates. These results and that of wild-type DGD suggest that hydrogen bonding with the substrate is required for decarboxylation. Mutants incapable of hydrogen bonding to the substrate, such as alanine, leucine and tryptophan mutants, showed negligible decarboxylation reactions. Transamination rates increased for some mutants and decreased for others. These data imply that the DGD subsite is influenced by the presence of glutamine 52. Furthermore, there is evidence showing that the subsite environment of wild-type DGD, the histidine and the glutamate mutants are different; the three DGD forms exhibited different chromophores at around $\rm\lambda\sb{max}$ of 500 nm when treated with 2-methylalanine or L-alanine in the presence of 3% glycerol. These results have important implications for other PLP-dependent enzymes, such as ornithine aminotransferase and $\gamma$-aminobutyrate aminotransferase. Since protein sequence alignment indicates DGD is homologous to the two aminotransferases, mutations at amino acid position corresponding to glutamine 52 of DGD at the active sites of these aminotransferases could disrupt the functionality of the enzymes. Protein sequence alignment showed that all but one of the PLP-dependent aminotransferases lack residues at position 52 capable of hydrogen bonding with the substrate carboxyl group, further re-affirming the role of glutamine 52 in decarboxylation.