79 - An Internal Sequence Region Catalytically Essential for the Human Selenophosphate Synthetase 2

Abstract

Mammalian homologues, Sephs1 and Sephs2, differ greatly in their ability to complement selD mutation in E. coli. Sephs2 is a selenoenyzme that has the Sec60 residue in the active site. Sephs2 retained the in vivo complementation when the Sec residue was replaced with Cys. Therefore, Sephs2Cys gene allowed the production of the bacterial selenoenzyme, formate dehydrogenases, in a selD mutant of E. coli. In contrast, Sephs1 has a Thr residue at the corresponding active site, and is an inactive catalysis producing no complementation effect in E. coli selD mutant. Although Sephs2 and Sephs1 share a high homology in their amino acid sequences, Sephs1-Thr29Cys mutant did not complement selD mutation in E. coli, suggesting that the lack of the active site Cys was not the sole reason for the inert catalysis. Sephs2 gene encodes an internal Pro-rich sequence spanning Glu43-Arg83 (designated as int2), while Sephs1 has a corresponding, but rather a shorter sequence Ala74-Ala114 (designated as int1). The in vivo complementation activity of Sephs2 was completely lost when the int2 sequence was substituted for the int1 sequence. Conversely, Sephs1 gained the SEPHS activity when the int1 region was replaced with int2 sequence in addition to the Thr to Cys substitution at the active site. Our results demonstrated for the first time that structural requirement of mammalian Sephs includes the proline-rich int2 region, although bacterial SEPHS including E. coli SelD has not such an internal sequence region corresponding to the int2 sequence. Interestingly, co-expression of Sephs1 gene under varied concentrations of L-arabinose lead to gradual suppression of selD complementation by Sephs2Cys gene in E. coli. The hypothesis that the inactive homologue Sephs1 can suppress Sephs2Cys activity is also supported by in vitro pull-down assay and yeast-2-hybrid assay, which both indicated binding of Sephs1 to Seph2Cys.