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Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme

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Abstract

Phosphorylation is an important pathway for the regulation of nitric oxide synthase (NOS) at the posttranslational level. However, the molecular underpinnings of NOS regulation by phosphorylations remain unclear to date, mainly because of the problems in making a good amount of active phospho-NOS proteins. Herein, we have established a system in which recombinant rat nNOS holoprotein can be produced with site-specific incorporation of phosphoserine (pSer) at residue 1412, using a specialized bacterial host strain for pSer incorporation. The pSer1412 nNOS protein demonstrates UV–Vis, far-UV CD and fluorescence spectral properties that are identical to those of nNOS overexpressed in other bacterial strains. The protein is also functional, possessing normal NO production and NADPH oxidation activities in the presence of abundant substrate l-Arg. Conversely, the rate of FMN–heme interdomain electron transfer (IET) in pSer1412 nNOS is considerably lower than that of wild-type (wt) nNOS, while the phosphomimetic S1142E mutant possesses similar electron transfer kinetics to that of wt. The successful incorporation and high yield of pSer1412 into rat nNOS and the significant change in the IET kinetics upon the phosphorylation demonstrate a highly useful method for incorporating native phosphorylation sites as a substantial improvement to commonly used phosphomimetics.

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Acknowledgements

We thank Dr. Charles Melancon for helpful discussions. This work was supported by the National Institutes of Health (Grant no. GM-081811 to CF, GM-057378 to MLK and GM-081568 to LR). Mass spectrometry data were acquired by the University of Ari-zona Analytical and Biological Mass Spectrometry Facility supported by NIH/NCI Grant CA023074 to the University of Arizona Cancer Center and by the BIO5 Institute of the University of Arizona.

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  1. Jingxuan He

    Present address: Department of Chemistry, Penn State University, University Park, PA, 16802, USA

Authors and Affiliations

  1. College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA

    Huayu Zheng, Jinghui Li & Changjian Feng

  2. Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM, 87131, USA

    Huayu Zheng, Jingxuan He, Jing Yang, Martin L. Kirk & Changjian Feng

  3. Department of Biochemistry and Structural Biology, University of Texas Health Science Center in San Antonio, San Antonio, TX, 78229, USA

    Linda J. Roman

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Correspondence to Changjian Feng.

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Zheng, H., He, J., Li, J. et al. Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme. J Biol Inorg Chem 24, 1–9 (2019). https://doi.org/10.1007/s00775-018-1621-1

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