title: Structure-based characterization of canine-human chimeric uricases and its evolutionary implications.
abstract: Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the β-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure-activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, PR China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.forename: Kai
authors.initials: K
authors.lastname: Fan
authors.forename: Weitao
authors.initials: W
authors.lastname: Zhang
authors.forename: Ruixin
authors.initials: R
authors.lastname: Zhu
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D004285:Dogs / Q000235:genetics*; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000737:chemistry*; D009154:Mutation; D010802:Phylogeny; D011323:Primates / Q000235:genetics; D016415:Sequence Alignment; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics*
chemical_list: D050506:Mutant Chimeric Proteins; D014503:Urate Oxidase