Adaptive evolution of multiple-variable exons and structural diversity of drug-metabolizing enzymes

Background: The human genome contains a large number of gene clusters with multiple-variablefirst exons, including the drug-metabolizing UDP glucuronosyltransferase ( UGT1) and 1- branching beta- 1,6-N-acetylglucosaminyltransferase (GCNT2, also known as IGNT) clusters, organized in a tandem array, similar to that of the protocadherin (PCDH), immunoglobulin ( IG), and T-cell receptor ( TCR) clusters. To gain insight into the evolutionary processes that may have shaped their diversity, we performed comprehensive comparative analyses for vertebrate multiple-variable-firstexon clusters. Results: We found that there are species-specific variable-exon duplications and mutations in the vertebrate Ugt1, Gcnt2, and Ugt2a clusters and that their variable and constant genomic organizations are conserved and vertebrate-specific. In addition, analyzing the complete repertoires of closely-related Ugt2 clusters in humans, mice, and rats revealed extensive lineage-specific duplications. In contrast to the Pcdh gene clusters, gene conversion does not play a predominant role in the evolution of the vertebrate Ugt1, Gcnt2 and Ugt2 gene clusters. Thus, their tremendous diversity is achieved through '' birth-and-death '' evolution. Comparative analyses and homologous modeling demonstrated that vertebrate UGT proteins have similar three-dimensional structures each with N- terminal and C-terminal Rossmann-fold domains binding acceptor and donor substrates, respectively. Molecular docking experiments identified key residues in donor and acceptor recognition and provided insight into the catalytic mechanism of UGT glucuronidation, suggesting the human UGT1A1 residue histidine 39 ( H39) as a general base and the residue aspartic acid 151 ( D151) as an important electron-transfer helper. In addition, we identified four hypervariable regions in the N- terminal Rossmann domain that form an acceptor-binding pocket. Finally, analyzing patterns of nonsynonymous and synonymous nucleotide substitutions identified codon sites that are subject to positive Darwinian selection at the molecular level. These diversified residues likely play an important role in recognition of myriad xenobiotics and endobiotics. Conclusion: Our results suggest that enormous diversity of vertebrate multiple variable first exons is achieved through '' birth-and-death '' evolution and that adaptive evolution of specific codon sites enhances vertebrate UGT diversity for defense against environmental agents. Our results also have interesting implications regarding the staggering molecular diversity required for chemical detoxification and drug clearance.