TY - JOUR
T1 - α-Amine Desaturation of d -Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l -Arginine 3-Hydroxylase, VioC
AU - Dunham, Noah P.
AU - Mitchell, Andrew J.
AU - Del Río Pantoja, José M.
AU - Krebs, Carsten
AU - Bollinger, J. Martin
AU - Boal, Amie K.
N1 - Funding Information:
*E-mail: ckrebs@psu.edu. *E-mail: jmb21@psu. *E-mail: akb20@psu.edu. ORCID Carsten Krebs: 0000-0002-3302-7053 J. Martin Bollinger, Jr.: 0000-0003-0751-8585 Amie K. Boal: 0000-0002-1234-8472 Present Addresses §A.J.M.: Whitehead Institute for Biomedical Research, Cambridge, MA 02142. ⊥J.M.D.R.-P.: Harvard University, Cambridge, MA 02138. Funding This work has been supported by the National Institutes of Health (GM119707 to A.K.B., GM127079 to C.K., and GM113106 to J.M.B. and C.K.) and the Searle Scholars Program (to A.K.B.). Notes The authors declare no competing financial interest.
Funding Information:
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corp. and the Michigan Technology Tri-Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The Eiger 16M detector was funded by an National Institutes of Health Office of Research Infrastructure Programs, High-End Instrumentation Grant (1S10OD012289-01A1).
Publisher Copyright:
© Copyright 2018 American Chemical Society.
PY - 2018/11/20
Y1 - 2018/11/20
N2 - When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.
AB - When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.
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U2 - 10.1021/acs.biochem.8b00901
DO - 10.1021/acs.biochem.8b00901
M3 - Article
C2 - 30403469
AN - SCOPUS:85056493724
VL - 57
SP - 6479
EP - 6488
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 46
ER -