Abstract
Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.
Highlights
Selenophosphate synthetase (SPS)1 from E. coli [3] and the closely related enzyme from Haemophilus influenzae [4] have been characterized
The attractive possibility that these lyases may serve normally to deliver Se0 to SPS was suggested by previous studies in which it was shown that L-selenocysteine and the Azotobacter vinelandii NifS protein effectively replaced the high level of free selenide in the in vitro SPS assay [9]
We report the ability of the CsdB, CSD, and in the presence of a lyase (IscS) proteins to function both in vitro and in vivo as selenium delivery proteins to the E. coli wild-type SPS and to SPS (C17S) mutant
Summary
Vol 275, No 31, Issue of August 4, pp. 23769 –23773, 2000 Printed in U.S.A. Escherichia coli NifS-like Proteins Provide Selenium in the Pathway for the Biosynthesis of Selenophosphate*. The attractive possibility that these lyases may serve normally to deliver Se0 to SPS was suggested by previous studies in which it was shown that L-selenocysteine and the Azotobacter vinelandii NifS protein effectively replaced the high level of free selenide in the in vitro SPS assay [9]. Selenium Delivery Activity of E. coli NifS-like Proteins proteins CsdB, CSD, and IscS revealed considerable differences in the degree of discrimination between L-cysteine and Lselenocysteine as substrates [12]. Mutagenesis of cysteine 17, located in the potential ATP-binding site, to serine resulted in the complete loss of detectable SPS activity in both the in vitro selenide-dependent assay and the in vivo complementation of a selD lesion in E. coli strain MBO8 [13]. We report the ability of the CsdB, CSD, and IscS proteins to function both in vitro and in vivo as selenium delivery proteins to the E. coli wild-type SPS and to SPS (C17S) mutant
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