A Remarkable Oxidative Cascade That Replaces the Riboflavin C8 Methyl with an Amino Group during Roseoflavin Biosynthesis

Article abstract of DOI:10.1021/jacs.6b02469

Roseoflavin is a naturally occurring riboflavin analogue with antibiotic properties. It is biosynthesized from riboflavin in a reaction involving replacement of the C8 methyl with a dimethylamino group. Herein we report the identification of a flavin-dependent enzyme that converts flavin mononucleotide (FMN) and glutamate to 8-amino-FMN via the intermediacy of 8-formyl-FMN. A mechanistic proposal for this remarkable transformation is proposed.

Full text of DOI:10.1021/jacs.6b02469

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Communication
A remarkable oxidative cascade that replaces the riboflavin C8
methyl with an amino group during roseoflavin biosynthesis
Isita Jhulki, Prem K. Chanani, Sameh Abdelwahed, and Tadhg P. Begley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b02469 • Publication Date (Web): 22 Jun 2016
Downloaded from http://pubs.acs.org on June 27, 2016
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A remarkable oxidative cascade that replaces the riboflavin C8 me-
thyl with an amino group during roseoflavin biosynthesis
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Isita Jhulki, Prem K. Chanani, Sameh Abdelwahed and Tadhg P. Begley*
Department of Chemistry, Texas A&M University, 3255 TAMU, College Station, TX 77843, USA
Supporting Information Placeholder
therefore proposed that Orf7989 might encode an 8-amino-
riboflavin synthase.
ABSTRACT: Roseoflavin is a naturally occurring riboflavin
analogue with antibiotic properties. It is biosynthesized from
riboflavin in a reaction involving replacement of the C8 me-
thyl with a dimethylamino group. Herein we report the iden-
tification of a flavin-dependent enzyme that converts flavin
mononucleotide (FMN) and glutamate to 8-amino-FMN via
the intermediacy of 8-formyl-FMN. A mechanistic proposal
for this remarkable transformation is proposed.
O
H
N
N
N
O
N
N
O
7M Methanolic ammonia
H
H
NH
NH
40oC, 12 hr
30% yield
N
4
N
5
O
O
Figure 2: Reaction of ammonia with 8-formyl-lumiflavin (4)
to form 8-amino-lumiflavin (5). This reaction is a model sys-
tem for the remarkable methyl group replacement by am-
monia that occurs during roseoflavin biosynthesis.
Orf7989 was overexpressed in E. coli BL21(DE3) and puri-
fied on a nickel NTA affinity column. The purified protein
was yellow, consistent with flavin binding. LC-MS analysis of
the metabolites that copurified with Orf7989 revealed the
presence of bound FMN, 8-formyl-FMN and 8-amino-FMN
(Figures 3&4). This observation suggested that Orf7989, het-
erologously expressed in the absence of all of the other ro-
seoflavin biosynthetic enzymes, catalyzed the formation of 8-
amino-FMN from FMN via the intermediacy of 8-formyl-
FMN (Figure 5). Even though it is generally assumed that
product dissociation rates are much faster than enzyme puri-
fication rates, we have previously found several other exam-
ples of enzymes that co-purify with product or intermediates
and view this type of analysis as one of the key experiments
for the identification of the reaction catalyzed by an enzyme
of unknown function.11-13
Roseoflavin (3, 8-dimethylamino-riboflavin), is a red flavin
antivitamin synthesized by Streptomyces davawensis and
Streptomyces cinnabarinus.1,2 It is the only biosynthesized
flavin analog with antibiotic activity and binds to the FMN
riboswitch, thus repressing riboflavin biosynthesis.3,4 Roseo-
flavin has been used in the screening of mutants for maxi-
mum riboflavin production because bacteria overproducing
riboflavin are resistant to roseoflavin toxicity.5,6 Remarkably,
roseoflavin is biosynthesized from riboflavin by replacing the
C8 methyl with a dimethylamino group (Figure 1).7,8 This is
an unprecedented transformation in flavoenzymology. The
methyltransferase catalyzing the final step of this transfor-
mation has been characterized9,10 and the biosynthetic gene
cluster from S. davawensis has been cloned and sequenced.2
The mechanism of the methyl group replacement chemistry
has not yet been elucidated and is the subject of this paper.
OH
OH
OH
HO
N
HO
N
HO
N
OH
OH
OH
OH
H
N
OH
Me
N
OH
N
O
N
O
N
O
RosA
?
H
Me
NH
NH
NH
N
1
N
3
N
2
O
O
O
Roseoflavin
Amino-riboflavin
Riboflavin
Figure 1: Roseoflavin biosynthesis from riboflavin via amino-
riboflavin in S. davawensis.
Based on a mechanistic model (vide infra), it was discov-
ered that 8-formyllumiflavin reacts with ammonia to form 8-
aminolumiflavin in 30% yield (Figure 2) (Jhulki, I. and
Begley, T. unpublished). This reaction suggests that roseofla-
vin might be formed from 8-formyl-riboflavin in a similar
oxidative displacement reaction.
Figure 3. Analysis of the orf7989 encoded protein. A) SDS-
PAGE gel of the purified enzyme (MW=29 kDa). B) UV-Vis
spectrum of Orf7989 showing an absorption maximum at
475 nm suggesting the presence of bound amino-FMN.
To test the hypothesis shown in Figure 5, we identified
formyl-FMN as the product formed when FMN is treated
with AFMN synthase in the absence of the amine donor
(Figures 6,7&8). This reaction is oxygen requiring and does
Examination of the genes in the sequenced roseoflavin bio-
synthetic cluster from S. davawensis revealed that only one
gene in this cluster was annotated as a flavoenzyme
(BN159_7989, NADPH-dependent FMN reductase). We
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not occur under anaerobic conditions. The search for a suit-
A mechanistic proposal for this remarkable conversion is
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able amine donor revealed that ammonia, pyridoxamine,
tyrosine, serine, histidine, tryptophan, phenylalanine, methi-
onine, arginine, lysine and aspartate were
shown in Figure 10. In this proposal deprotonation of the C8
methyl group of FMN followed by oxidation gives the hy-
droperoxide 10.14 The C8 methyl group is known to be acidic
and this chemistry is also thought to occur during the cova-
lent attachment of flavins to active site histidine and cysteine
residues.15-17 Next, an
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Figure 4. Analysis of the denatured Orf7989 encoded enzyme showing that the protein co-purified with amino-FMN, formyl-FMN
and FMN. A) Extracted ion chromatogram (EIC) at m/z 458.1077. B) ESI-MS of the 458.1077 Da species consistent with amino-
FMN. C) EIC at m/z 471.0917. D) ESI-MS of the 471.0917 Da species consistent with formyl-FMN. E) EIC at m/z 457.1124. F) ESI-MS
of the 457.1124 Da species consistent with FMN. All spectra were obtained in positive ion mode.
not donors, glutamine was a weak donor and glutamate was
the best donor (Figure 6&7) and was converted to α-
ketoglutarate (Figure 9).
active site base abstracts a C8 hydrogen to cleave the perox-
ide, leading to the formation of 8-formyl-FMN 7. Amine ad-
dition to C8 followed by deformylation and air oxidation
gives 21. Two successive tautomerizations give 23. Imine hy-
drolysis and air oxidation completes the formation of 8-
R
N
O
R
N
R
N
Ammonia
donor
N
O
N
O
O
H2N
N
O
O
H
NH
NH
NH
N
6
N
N
O
7
8
R= Ribose phosphate
Figure 5: Proposal for the amino-FMN (AFMN) synthase-
catalyzed conversion of FMN to AFMN.
Figure 6. AFMN synthase-catalyzes the formation of formyl-
FMN and amino-FMN in the absence and presence of
glutamate. A) HPLC analysis of the AFMN synthase-
catalyzed oxidation of FMN followed by derivatization with
pentafluorobenzyl hydroxylamine (PFBHA, blue trace, no
glutamate and red trace, no enzyme control). The peaks
eluting after 32.2 and 33 min correspond to the two PFBHA
oxime isomers. B) HPLC analysis of the AFMN synthase-
catalyzed oxidation of FMN in the presence of glutamate
(blue trace, full reaction and red trace, no enzyme conrol).
amino-FMN 8.
Figure 7. Co-elution experiments with enzymatically formed
formyl-FMN and amino-FMN and synthesized standards. A)
HPLC analysis of enzymatically formed amino-FMN (blue
trace-enzymatically formed amino-FMN, red trace-amino-
FMN standard, and green trace-co-elution of standard and
enzymatic reaction mixture). B) HPLC analysis of
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enzymatically formed formyl-FMN derivatized with
pentafluorobenzyl hydroxylamine (blue trace-enzymatically
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formed formyl-FMN (absence of glutamate) red trace-formyl-
FMN PFBHA oxime standard, and green trace-co-elution of
standard and enzymatic reaction mixture after treatment
with pentafluorobenzyl hydroxylamine). Diagrams C) and D)
represent the same set of samples after phosphatase
treatment showing comigration of amino-riboflavin and
formyl-riboflavin PFBHA oxime with the dephosphorylated
enzymatic products derivatized with PFBHA.
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Figure 9. LC-MS analysis of the α-ketoglutarate (24) PFBHA
oxime generated from glutamate (17) in the AFMN synthase
catalyzed reaction (negative mode). Left- Extracted Ion
Chromatogram (EIC) at m/z 340.0244 for the PFBHA trapped
α-ketoglutarate (red trace- trapped α-KG formed in the en-
zymatic reaction, blue trace- trapped α-KG standard and
green trace- co-elution of trapped α-KG standard and same
formed in the AFMN synthase catalyzed reaction. Right- ESI-
MS of the 340.0244 Da species consistent with the ketoglu-
tarate PFBHA oxime.
Figure 8. Comparison of the UV-Visible spectra of formyl and
amino-FMN generated in the AFMN synthase-catalyzed reac-
tion with authentic standards. A) Overlay of the UV-Visible
spectra of enzymatically formed formyl-FMN (7, Blue trace),
formyl-lumiflavin (4, Red trace) and FMN (6, Green trace). B)
Overlay of the UV-Visible spectra of enzymatically formed
amino-FMN (8, Green trace), formyl-FMN (7, Red trace),
amino-lumiflavin (5, Blue trace).
Thus, what initially appeared to be a highly complex reac-
tion, requiring several enzymes, upon scrutiny turns out to
be quite simple, involving an initial oxidation of the C8 me-
thyl group to formyl-FMN followed by a 4-electron oxidative
cascade18-21 in which electrons are iteratively removed using
the well-precedented oxidation of dihydroflavin by molecular
oxygen. This study underscores the power of mechanistic
reasoning to identify gene function. Mechanistic and struc-
tural studies are in progress to elucidate the details of this
fascinating new riboflavin transfor-mation.
This mechanistic proposal is consistent with the
observed substrates and products of the reaction and with
the intermediacy of formyl-FMN and with the model system
developed for the conversion of formyl-FMN to amino-FMN
(Figure 2). We have also demonstrated that AFMN synthase
catalyzes the deprotonation of the C8 methyl group of FMN
(Figure S7) and that glutamic acid, the nitrogen source (la-
beling study in SI, Figure S4), is converted to α-ketoglutarate
(Figure 9). No flavin modification is detected when the reac-
tion is run in the absence of oxygen and glutamic acid, sug-
gesting that 8-hydroxymethyl-FMN (13) is not an intermedi-
ate. This is further supported by demonstrating that hydro-
gen peroxide is not formed in the AFMN synthase catalyzed
oxidation of FMN to formyl-FMN (Figure S5 & S6).
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ASSOCIATED CONTENT
Supporting Information
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Detailed experimental procedures for the syntheses and
characterization of formyl-lumiflavin (4), formyl-FMN (7),
amino-FMN (8) and the PFBHA derivatized oxime standards,
AFMN synthase overexpression and purification, enzymatic
assays, H/D exchange experiment, co-elution experiment,
measurement of oxygen consumption for formyl-FMN for-
mation in AFMN synthase assay, NMR and LCMS analysis.
AUTHOR INFORMATION
Corresponding Author
begley@chem.tamu.edu
Note
While this paper was under review, similar findings were
reported by Professor Mack using a genetics rather than a
chemical approach to the identification of AFMN synthase.22
The papers differ in some of the details of the reconstitution
and mechanistic analysis.
ACKNOWLEDGMENT
This research was supported by the Robert A. Welch Founda-
tion (A-0034 to TPB) and by a grant from the National Insti-
tutes of Health (DK44083).
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