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#-Amine desaturation of D-arginine by the iron(II)- and 2(oxo)-glutarate-dependent L-arginine 3-hydroxylase, VioC Noah P. Dunham, Andrew J. Mitchell, José Del Río-Pantoja, Carsten Krebs, J. Martin Bollinger, Jr., and Amie K. Boal Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00901 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018
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Biochemistry
-Amine Desaturation of D-Arginine by the Iron(II)and 2-(Oxo)-glutarate-dependent L-Arginine 3Hydroxylase, VioC Noah P. Dunham,1 Andrew J. Mitchell,1† José M. Del Río Pantoja,1 Carsten Krebs,1,2* J. Martin Bollinger Jr.,1,2* and Amie K. Boal1,2*
1Department
of Biochemistry and Molecular Biology and 2Department of
Chemistry, The Pennsylvania State University, University Park PA 16802
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ABSTRACT
When challenged with substrate analogs, 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 more likely,
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Biochemistry
because the former mechanism would be expected to result in detectable incorporation from
18O
2.
18O
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 4,5desaturase, 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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INTRODUCTION
The twenty standard amino acids serve as important building blocks in biology, acting as both the constituents of macromolecular protein polymers and platforms for synthesis of small-molecule metabolites. In either role, specific modifications by enzymes provide additional biochemical diversity critical for function.1 The iron(II)- and 2-(oxo)-glutarate-dependent (Fe/2OG) oxygenase superfamily is responsible for a large fraction of the known oxidative amino acid
modifications.2
In
eukaryotes,
amino-acid-targeting
Fe/2OG
enzymes
hydroxylate side chain functional groups and operate predominantly on protein or peptide substrates for structural or regulatory functions.3,4 In prokaryotes, Fe/2OG catalysts can additionally target monomeric amino acids, and many different reaction outcomes are possible.5 A classic example is clavaminate synthase
(CAS),
which
performs
sequential
hydroxylation,
cyclization,
and
desaturation reactions on an L-arginine (L-Arg) derivative in the biosynthesis of the -lactamase inhibitor, clavulanic acid.6 Fe/2OG enzymes that utilize amino acid substrates often exhibit considerable variability in regiochemistry as well.
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For example, the enzymes VioC, NapI, and OrfP all act on L-Arg but, despite sharing significant sequence similarity (~ 50%), target different positions of the amino acid side chain toward the biosynthesis of distinct natural products: VioC is a 3-hydroxylase in the viomycin pathway,7,8 OrfP is a 3,4-dihydroxylase in streptothricin pathways,9 and NapI is a 4,5-desaturase in the naphthyridinomycin pathway.10,11 Examples of Fe/2OG enzymes that transform many other amino acids
are
known,
proteinogenic
but,
to
L-enantiomer.
date,
they
Non-native
all
target
reactivity
the
side
with
the
chain
of
the
opposite
(D)
enantiomer has not, to our knowledge, been investigated.
Most Fe/2OG enzymes share a HXD/EXnH Fe(II) binding motif (known as the facial triad12,13) and initial steps of catalysis, regardless of their substrate or reaction type.14 Binding of the primary substrate triggers dissociation of the remaining water ligand15 and addition of O2 to the open coordination site of the Fe(II) cofactor.16 The non-coordinating O-atom of the resulting Fe(III)–superoxo intermediate is transferred (in multiple steps) to C2 of the 2OG substrate, converting it to CO2 and succinate, while the coordinated O-atom becomes the
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oxo
ligand
of
the
substrate-targeting
Fe(IV)–oxo
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(ferryl)
intermediate.
In
transformations of aliphatic sites, the ferryl complex abstracts hydrogen (H•) from the substrate. Its potency typically enables activation of even very inert carbon centers. The resultant state, containing a carbon-centered substrate radical (C•) and Fe(III)–OH cofactor, is an important branch point: how it reacts in ensuing steps dictates the reaction outcome. In hydroxylations, the C• attacks the oxygen of the Fe(III)–OH complex, forming a new C–O bond and regenerating the Fe(II) cofactor for subsequent turnover. This radical-coupling step, termed oxygen rebound,17 is thought to have a low activation barrier, consistent with the failure of the C•/Fe(III)–OH state to have been detected in transient-kinetic analyses of Fe/2OG hydroxylases.
In reaction outcomes other than hydroxylation, the C•/Fe(III)–OH state often has a different fate. Oxygen rebound can be almost completely suppressed, likely enabled in at least some enzymes by a different geometric structure of the ferryl complex. For example, in the Fe/2OG aliphatic halogenases, a ciscoordinated chloride or bromide ligand is transferred to the substrate radical in
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Biochemistry
preference to oxygen rebound.18-22 For the chlorinases SyrB2 and WelO5 from the syringomycin and welwitindolinone biosynthetic pathways (respectively), it is thought that the alternative radical-coupling step is enforced by an unusual disposition of the C–H and Fe=O bonds, achieved by a ~ 90° relocation of the oxo ligand to an off-line position in the key ferryl complex.20,23-26 Such ligand reorganization could be a common strategy for suppression of rebound in Fe/2OG enzymes with other non-canonical (non-hydroxylation) reactivities.
Scheme 1. Possible mechanisms by which hydroxylated intermediates produced in reactions of Fe/2OG oxygenases can be further processed to yield alternative outcomes.
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Outcomes other than hydroxylation may also arise by pathways in which the facile rebound step does occur but the hydroxylated species then undergoes further processing. For example, a number of prokaryotic and eukaryotic Fe/2OG N-demethylases implicated in gene regulation and DNA repair (AlkB, ALKBH1-8, FTO, and histone demethylases) are proposed to exploit initial hydroxylation followed by fragmentation of the unstable hemiaminal intermediate to the corresponding amine and formaldehyde (Scheme 1A).27-32 Similarly, mechanisms involving transient C–O bond formation have been considered for a
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range of desaturation reactions by Fe/2OG enzymes. In these systems, the installed hydroxyl group would subsequently be jettisoned, either in an heteroatom-assisted dehydration (Scheme 1B) or as nucleofuge in a Grob-type fragmentation (Scheme 1C), with formation of a new C=X (X= N, O) or C=C double
bond,
hydroxylated
respectively.33-35 product
is
Although
certainly
spontaneous
plausible
in
the
breakdown
demethylase
of
the
reactions,
enzymatic assistance after hydroxylation would be required in the desaturation reactions.
The aforementioned Fe/2OG L-Arg 3-hydroxylase, VioC, has recently been used as a platform for crystallographic characterization of reaction cycle intermediates and their stable mimics.36 The enzyme can also accept alternative substrates and, in one case, was shown to enable a non-native outcome with such an analog. Reaction of VioC with L-homoarginine (L-hArg, the L-Arg analog with an additional methylene unit in its side chain) resulted in a 3,4desaturation
outcome
in
Comparative
mechanistic
competition analysis
of
with
hydroxylation
of
this
reaction
the
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and
either native
site.33 4,5-
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desaturation
of
L-Arg
by
NapI
showed
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that
they
proceed
by
different
mechanisms. Whereas NapI mediates transient C5–N6 desaturation and C4–H cleavage by deprotonation (Scheme S1A), the absence of a heteroatom in the 3,4-desaturation of L-hArg by VioC necessitates a less selective mechanism involving
sequential
HAT
steps
[to
the
ferryl
and
resultant
Fe(III)–OH
intermediates] (Scheme S1B). Here, we report discovery and analysis of a second non-native desaturation by VioC in our ongoing efforts to map and rationalize the enzyme family’s full range of catalytic capabilities. Specifically, we show that the native 3-hydroxylase readily binds and transforms the Denantiomer of its native L-Arg substrate to the corresponding 2-ketoacid product in an oxidative deamination reaction (Scheme 2). The simplest possible adaptation
of
the
native
reaction
mechanism
that
would
rationalize
the
alternative outcome — C2 hydroxylation and subsequent deamination — would be reminiscent of the mechanism purported for the Fe/2OG N-demethylases. However, the results of in-depth analysis of the reaction by isotope-tracer and chemical-trapping experiments show that the reaction actually proceeds through
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Biochemistry
an iminium intermediate that subsequently undergoes hydrolysis. The oxidative deamination of D-Arg thus appears to proceed by α-amine desaturation, a pathway with direct analogy to the initial steps in the proposed mechanism of olefin installation by NapI.33
Scheme 2. VioC reactions with L-Arg and D-Arg.
EXPERIMENTAL DETAILS
General
Methods.
Liquid
chromatography-mass
spectrometry
(LC-MS)
experiments were carried out on an Agilent 1260 series LC system interfaced with an Agilent 6460 triple-quadrupole mass spectrometer. All reagents were used directly as obtained from the commercial sources. The relative molecular mass and purity of enzyme samples were determined by SDS-polyacrylamide
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gel electrophoresis (SDS-PAGE). Crystallographic (Table S1) and spectroscopic methods are described in the Supporting Information.
Reactions of VioC with D-arginine. Assays were prepared in a Lab Master 100 anoxic chamber (MBraun) in a total volume of 200 µL in 100 mM Tris-HCl buffer, pH 7.5 (reaction buffer). The final concentrations were 10 µM VioC, 10 µM (NH4)2Fe(SO4)2, 0.8 mM 2OG, and 1 mM D-arginine. Concentrated reaction mixtures were removed from the anoxic chamber and the reaction initiated by diluting to the final volume with cold air-saturated reaction buffer. Tubes were then opened to air and stirred gently, and the reaction was allowed to proceed for ~1 h. A small quantity of NaBH4 (
