Increased Catalytic Efficiency following Gene Fusion of Bifunctional Methionine Sulfoxide Reductase Enzymes from Shewanella oneidensis

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• 作者：Baowei Chen ; Lye Meng Markillie ; Yijia Xiong ; M. Uljana Mayer ; Thomas C. Squier
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 11, 2007
• 年：2007
• 卷：46
• 期：49
• 页码：14153 - 14161
• 全文大小：438K
• 年卷期：v.46,no.49(December 11, 2007)
• ISSN：1520-4995

文摘

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and togetherfunction as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes arefused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate howgene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressedthe MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. Forcomparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a geneticallyengineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatoryprotein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymestogether at 25 C. A restoration of the normal protein fold is observed co-incident with the repair ofMetSO in oxidized CaM (CaM_ox by MsrBA, as monitored by time-dependent increases in the anisotropyassociated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein(FlAsH). Underlying the efficient repair of MetSO in CaM_ox is the coordinate activity of the two catalyticdomains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement incomparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of bothdomains of MsrBA permits the full repair of all MetSO in CaM_ox. The common expression of Msr fusionproteins in bacterial pathogens is consistent with an important role for this enzyme activity in themaintenance of protein function necessary for bacterial survival under highly oxidizing conditions associatedwith pathogenesis or bioremediation.