Structural and kinetic studies on RosA, the enzyme catalysing the methylation of 8-demethyl-8-amino- d -riboflavin to the antibiotic roseoflavin

Article abstract of DOI:10.1111/febs.13690

N,N-8-demethyl-8-amino-d-riboflavin dimethyltransferase (RosA) catalyses the final dimethylation of 8-demethyl-8-amino-d-riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study, we solved the X-ray structure of RosA, and determined the binding properties of substrates and products. Moreover, we used steady-state and rapid reaction kinetic studies to obtain detailed information on the reaction mechanism. The structure of RosA was found to be similar to that of previously described S-adenosylmethionine (SAM)-dependent methyltransferases, featuring two domains: a mainly α-helical 'orthogonal bundle' and a Rossmann-like domain (α/β twisted open sheet). Bioinformatics studies and molecular modelling enabled us to predict the potential SAM and AF binding sites in RosA, suggesting that both substrates, AF and SAM, bind independently to their respective binding pocket. This finding was confirmed by kinetic experiments that demonstrated a random-order 'bi-bi' reaction mechanism. Furthermore, we determined the dissociation constants for substrates and products by either isothermal titration calorimetry or UV/Vis absorption spectroscopy, revealing that both products, RoF and S-adenosylhomocysteine (SAH), bind more tightly to RosA compared with the substrates, AF and SAM. This suggests that RosA may contribute to roseoflavin resistance in S. davawensis. The tighter binding of products is also reflected by the results of inhibition experiments, in which RoF and SAH behave as competitive inhibitors for AF and SAM, respectively. We also showed that formation of a ternary complex of RosA, RoF and SAH (or SAM) leads to drastic spectral changes that are indicative of a hydrophobic environment. Database Structural data are available in the Protein Data Bank under accession number 4D7K. 8-demethyl-N,N-8-amino-D-riboflavin dimethyltransferase (RosA) catalyzes the dimethylation of 8-demethyl-8-amino-D-riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis via a random-order mechanism. RosA has a structural topology similar to other S-adenosylmethionine (SAM)-dependent methyltransferases. Slow synthesis of RoF and the tight binding of the products, RoF and SAH, suggest that RosA may contribute to a mechanism for RoF resistance.

Full text of DOI:10.1111/febs.13690

Structural and kinetic studies on RosA, the enzyme
catalysing the methylation of 8-demethyl-8-amino-
D-riboflavin to the antibiotic roseoflavin
Chanakan Tongsook1,†, Michael K. Uhl2,†, Frank Jankowitsch3, Matthias Mack3, Karl Gruber2 and
Peter Macheroux1
1 Institute of Biochemistry, Graz University of Technology, Austria
2 Institute of Molecular Biosciences, University of Graz, Austria
3 Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, Germany
Keywords
N,N-8-demethyl-8-amino-D-riboflavin dimethyltransferase (RosA) catalyses
the final dimethylation of 8-demethyl-8-amino-D-riboflavin (AF) to the
antibiotic; crystallography; enzyme kinetics;
S-adenosylmethionine; transferase
antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present
study, we solved the X-ray structure of RosA, and determined the binding
properties of substrates and products. Moreover, we used steady-state and
Correspondence
K. Gruber, Institute of Molecular
rapid reaction kinetic studies to obtain detailed information on the reaction
mechanism. The structure of RosA was found to be similar to that of pre-
viously described S-adenosylmethionine (SAM)-dependent methyltrans-
ferases, featuring two domains: a mainly a-helical ‘orthogonal bundle’ and
a Rossmann-like domain (a/b twisted open sheet). Bioinformatics studies
and molecular modelling enabled us to predict the potential SAM and AF
binding sites in RosA, suggesting that both substrates, AF and SAM, bind
independently to their respective binding pocket. This finding was con-
firmed by kinetic experiments that demonstrated a random-order ‘bi-bi’
reaction mechanism. Furthermore, we determined the dissociation con-
stants for substrates and products by either isothermal titration calorimetry
or UV/Vis absorption spectroscopy, revealing that both products, RoF and
S-adenosylhomocysteine (SAH), bind more tightly to RosA compared with
the substrates, AF and SAM. This suggests that RosA may contribute to
roseoflavin resistance in S. davawensis. The tighter binding of products is
also reflected by the results of inhibition experiments, in which RoF and
SAH behave as competitive inhibitors for AF and SAM, respectively. We
also showed that formation of a ternary complex of RosA, RoF and SAH
(or SAM) leads to drastic spectral changes that are indicative of a
hydrophobic environment.
Biosciences, University of Graz,
Humboldstraße 50/3, A-8010 Graz, Austria
Fax: +43 316 380 9897
Tel: +43 316 380 5483
E-mail: karl.gruber@uni-graz.at
P. Macheroux, Institute of Biochemistry,
Graz University of Technology,
Petersgasse 12, A-8010 Graz, Austria
Fax: +43 316 873 6952
Tel: +43 316 873 6450
E-mail: peter.macheroux@tugraz.at
†These authors contributed equally to this
work.
(Received 7 December 2015, revised 26
January 2016, accepted 18 February 2016)
doi:10.1111/febs.13690
Database
Structural data are available in the Protein Data Bank under accession number 4D7K.
Abbreviations
AF, 8-demethyl-8-amino-D-riboflavin; ITC, isothermal titration calorimetry; MAD, multiple-wavelength anomalous dispersion; MAF,
8-demethyl-N-8-methylamino-D-riboflavin; RoF, roseoflavin; RosA, 8-demethyl-N,N-8-amino-D-riboflavin dimethyltransferase; SAH,
S-adenosylhomocysteine; SAM, S-adenosylmethionine.
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1
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

Structure and mechanism of RosA
C. Tongsook et al.
enzyme, with a turnover rate (kcat) of 0.06 minꢀ1 for
the overall dimethylation reaction. As RoF is a more
potent antibiotic than AF and MAF, the slow synthe-
sis of RoF and the tight binding of products may con-
tribute to protection of the strains S. davawensis and
S. cinnabarinus from the antibiotic that they produce.
Introduction
The Gram-positive soil bacteria Streptomyces davawen-
sis and Streptomyces cinnabarinus produce roseoflavin
(RoF), the only known natural riboflavin (vitamin B2)
analogue with antibiotic activity. In contrast to most
other antibiotics, RoF has multiple cellular targets and
negatively affects flavoproteins as well as FMN ribos-
witches [1–8]. RoF may be considered a natural anti-
metabolite, and was postulated to be biosynthesized
from riboflavin through 8-demethyl-8-amino-D-ribofla-
vin (AF) and 8-demethyl-8-methylamino-riboflavin
(MAF) [9,10]. The occurrence of the intermediates AF
and MAF was confirmed by the discovery of the first
enzyme of RoF biosynthesis, the S-adenosylmethionine
(SAM)-dependent dimethyltransferase N,N-8-demethyl-
8-amino-D-riboflavin dimethyltransferase (RosA) [11].
This enzyme synthesizes RoF from AF (via MAF) in
two consecutive methylation reactions, as shown in
Scheme 1. The corresponding gene (rosA) is present
in a cluster comprising ten genes. The remaining genes
of this cluster were found not to be involved in RoF
synthesis, and it is presently unclear which genes/en-
zymes are responsible for synthesis of the key interme-
diate AF [12].
Results
Crystal structure of RosA
Initial attempts to solve the structure of RosA by
molecular replacement were unsuccessful despite a
thorough search for appropriate template structures
using the online tools PHYRE2 [13], FUGUE [14] and
CASPR [15]. Therefore, RosA labelled with selenome-
thionine (SeMet) (seven methionine residues in 353
amino acids) was produced, and the structure was
solved by multiple-wavelength anomalous dispersion
(MAD) using a single tetragonal crystal (Table 1). The
structure (one protomer in the asymmetric unit) was
ꢀ
partially refined to a resolution of 3.5 A, and this
model was then used to solve the structure of a tri-
clinic crystal form (space group P1) by molecular
replacement. This crystal contained six molecules per
asymmetric unit, and the structure was refined at a res-
The important role of RosA for completion of RoF
biosynthesis prompted us to determine the three-
dimensional structure of this enzyme and to investigate
the mechanism of the consecutive methylation reac-
tions in more detail. We show that RosA has a
topology similar to other SAM-dependent methyltrans-
ferases, containing a mainly a-helical ‘orthogonal bun-
dle domain’ and a Rossmann-like fold. Determination
of the three-dimensional structure also enabled us to
locate the binding sites for the substrates AF and
SAM. In addition, we performed steady-state and
rapid reaction studies, and determined the dissociation
constants for the substrates of RosA as well as for the
products. It was found that AF and SAM bind inde-
pendently to RosA according to a random-order
mechanism. The products of the RosA reaction, RoF
and SAH, exhibit higher affinity for the protein than
the substrates AF and SAM do, leading to competitive
ꢀ
olution of 2.2 A, yielding final values of R = 20% and
Rfree = 25% (Table 2).
The RosA protomer comprises two domains: a
mainly a-helical ‘orthogonal bundle domain’ and a
Rossmann-like domain (a/b twisted open sheet). The
orthogonal bundle (five a-helices and one antiparallel
b-sheet) consists of residues 1–98. Residues 99–178
form an inter-domain region (five helices) that con-
nects the Rossmann motif to the orthogonal bundle.
The Rossmann motif (residues 179–353) comprises a
seven-stranded b-sheet core consisting of five parallel
and two antiparallel b-strands, which are connected by
two pairs of a-helices (Fig. 1A). To classify the
domains, we performed
a CATH database (http://
www.cathdb.info/) search using the CATHEDRAL
algorithm [16]. The orthogonal bundle belongs to the
CATH superfamily ‘winged helix repressor DNA bind-
ing domain’ (CATH classification: 1.10.10.10; Sequen-
product inhibition. RosA is
a
comparably slow
Ribityl
N
Ribityl
N
Ribityl
N
SAM
SAH
HN
SAM
SAH
N
N
O
N
O
O
H2N
N
O
O
O
NH
NH
NH
RosA
RosA
N
N
N
Scheme 1. The catalytic reaction of RosA.
2
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C. Tongsook et al.
Structure and mechanism of RosA
Table 1. Statistics for the SeMet MAD datasets. Values in parentheses are for the highest-resolution shell.
Peak
Inflection
Remote
X-ray source
SLS X06DA-PXIII
0.9791
ꢀ
Wavelength (A)
Temperature (K)
Space group
0.9796
0.9714
100
100
100
I4122
I4122
I4122
Cell dimensions
ꢀ
a = b, c (A)
112.54, 132.27
47.04–3.35 (3.53–3.35)
166 043 (22 355)
6400 (905)
112.46, 132.48
47.02–3.54 (3.60–3.54)
141 532 (20 491)
5454 (771)
112.60, 132.27
47.06–3.42 (3.73–3.42)
156 742 (21 751)
6031 (848)
ꢀ
Resolution (A)
Total number of reflections
Unique number of reflections
Multiplicity
25.9 (27.7)
26.0 (26.6)
26.0 (25.6)
Anomalous multiplicity
Completeness (%)
Anomalous completeness (%)
Rp.i.m. (%)
14.2 (13.1)
14.2 (14.1)
14.2 (13.7)
99.9 (99.7)
100.0 (100.0)
99.9 (99.4)
99.9 (99.6)
99.9 (99.4)
99.9 (99.3)
1.3 (5.9)
1.0 (5.7)
1.1 (6.3)
Rmeas (%)
6.4 (29.5)
5.2 (29.8)
5.6 (32.2)
<I/aI)>
CC1/2
48.3 (12.8)
51.1 (14.8)
50.0 (12.2)
1.000 (0.989)
1.000 (0.995)
1.000 (0.987)
1.000 (0.995)
1.000 (0.989)
1.000 (0.997)
CC*
tial Structure Alignment Program (SSAP) score = 87.6;
RMSD = 2.7 A) and the functional family ‘caffeic acid
3-O-methyltransferase 1’. The Rossmann-like domain
was found to be a member of the CATH superfamily
‘Vaccinia Virus protein VP39’ (CATH classification:
Z scores were mitomycin 7-O-methyltransferase from
Streptomyces lavendulae (PDB ID 3GWZ), a probable
phenazine-specific methyltransferase from Pseu-
domonas aeruginosa (PDB ID 2IP2), the CALO1
methyltransferase from Micromonospora echinospora
(PDB ID 3LST), and a caffeic acid O-methyltransfer-
ase from Sorghum bicolor (PDB ID 4PGH), with
Z scores of 33, 32, 30 and 30, respectively. These pro-
teins share 21–35% sequence identity with RosA.
Superposition with the most similar structure (3GWZ,
35% sequence identity, 76% query coverage) resulted
ꢀ
ꢀ
3.40.50.150; SSAP score = 85.4, RMSD = 4.1 A). The
algorithm treated the inter-domain residues as part of
the Rossmann motif.
A PISA analysis [17] predicted that a dimer is the
most probable oligomer of RosA in solution. This pre-
diction was confirmed by size exclusion chromatogra-
phy (Fig. 2). Dimerization occurs in a head-to-head
arrangement (Fig. 1B), and is mediated by interactions
of adjacent a-helices of the bundle motif (residues 1–36
and residues 60–73), the inter-domain a-helices com-
prising residues 101–109 and residues 111–126, and one
helix from the Rossmann motif (residues 293–305).
The RosA protomers in the asymmetric unit exhibit
very similar structures. Pairwise superpositions of the
Rossmann domains in the six crystallographically inde-
pendent protomers yielded RMSD values in the range
ꢀ
in an RMSD of 3.5 A for the isolated subunit (317 of
ꢀ
324 aligned Ca atoms) and 6.6 A for the dimers (618
of 650 aligned Ca atoms). Superposition of the Ross-
mann domains of RosA (residues 179–345) and 3GWZ
ꢀ
(residues 179–349) resulted in an RMSD of 0.9 A (118
of 160 aligned Ca atoms). This indicates that, despite
an overall low sequence identity, the structure of
RosA, especially the Rossmann domain, closely resem-
bles that of other methyltransferases.
ꢀ
from 0.2 to 0.9 A (for approximately 160 aligned Ca
atoms). The same analysis yielded mean RMSD values
Prediction of the SAM/SAH binding site
ꢀ
of 0.3 and 0.6 A for the N-terminal and intermediate
regions. The values are slightly higher for superposi-
ꢀ
tions of the entire chains (1.2 A) and the three dimers
Unfortunately, all efforts to co-crystallize RosA with
SAM, SAH, AF or combinations of these compounds
and cofactors were unsuccessful. Therefore, we used
structural bioinformatics methods to locate putative
binding sites for the substrate and cofactor. A cavity
analysis using the program CASOX [19] yielded several
cavities, but only one of those was large enough to
ꢀ
(1.6 A).
To identify protein structures resembling RosA in
terms of its tertiary structure, we performed a Dali ser-
ver analysis [18]. The structures with the highest
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3

Structure and mechanism of RosA
C. Tongsook et al.
Table 2. Data collection and refinement statistics. Values in
dicted 13 consensus SAH/SAM binding residues with
a C-score of 0.54 and a cluster size (total number of
templates in a cluster) of 103. Moreover, COACH gener-
ated a structure of RosA with SAM bound in the pre-
dicted active site. All other ligand–enzyme complexes
had significantly lower C-scores. Among those struc-
tures with C-scores between 0.5 and 0.05 were those
with ligands such as N-acetyl serotonin (C-score 0.10),
pisatin (C-score 0.08) and 5-(3,3-dihydroxypropeny)-3-
methoxy-benzene-1,2-diol (C-score 0.05). Flavin
derivatives such as AF did not occur in these clusters
identified by COACH. However, a cluster with an even
lower C-score of 0.02 contained structures of methyl-
transferases with bound SAH and 4-methoxy-E-rhodo-
mycin. 4-methoxy-E-rhodomycin resembles AF with
regard to the planar ring system. A comparison of the
modelled RosA–SAM complex with the structure of
carminomycin 4-O-methyltransferase from Strepto-
myces peucetius (PDB ID 1TW2), a representative of
the 4-methoxy-E-rhodomycin cluster, showed that,
despite the low sequence identity of 33%, the Ross-
mann domain and especially the b-sheet core appeared
to be structurally conserved. Therefore, we superim-
posed the b-sheet core residues of RosA and 1TW2
parentheses are for the highest-resolution shell.
RosA (native)
Data collection
X-ray source
ESRF ID23-1
1.000
ꢀ
Wavelength (A)
Temperature (K)
Space group
100 K
P1
Cell dimensions
ꢀ
a, b, c (A)
82.72, 82.76, 96.45
95.1, 98.7, 114.5
34.77–2.22 (2.26–2.22)
168 833 (3484)
86 780 (1838)
1.9 (1.9)
a, b, c (°)
Resolution (A)
ꢀ
Total number of reflections
Unique number of reflections
Multiplicity
Completeness (%)
Rp.i.m. (%)
Rmeas (%)
<I/r(I)>
77.4 (33.0)
3.1 (39.8)
4.3 (56.2)
12.7 (1.70)
CC1/2
0.999 (0.756)
1.000 (0.928)
CC*
Refinement
Rwork
0.1949
0.2485
15 787
15 518
269
Rfree
Number of atoms
Protein
ꢀ
Water
(RMSD 0.805 A; 42 of 42 aligned Ca atoms), and
2
ꢀ
B-factors (A )
aligned a molecule of AF with 4-methoxy-E-rhodomy-
cin bound to 1TW2 (Fig. 4A). The RosA–AF–SAM
complex thus generated was energy-minimized and
analysed in terms of the binding interactions of AF
and SAM (Figs 3B and 4B).
Protein
Water
66.1
48.9
65.8
All atoms
RMSDs
ꢀ
Bond lengths (A)
0.003
0.620
0.15
Bond angles (°)
Ramachandran outliers (%)
PDB ID
Determination of dissociation constants
4D7K
Inspection of the AF and SAM binding sites in the
RosA structure suggested that both substrates, AF
and SAM, bind independently to their respective pock-
ets. Thus we determined the dissociation constant for
AF and SAM by isothermal titration calorimetry
(ITC). As shown in Fig. 5A,C, titration with either
AF or SAM produced exothermic signals that were fit-
ted to a one-binding-site model, yielding dissociation
constants of 10 ꢁ 1 and 22 ꢁ 2 lM, respectively. Sur-
prisingly, both products, i.e. RoF and SAH, bind
approximately ten times more tightly than the sub-
strates, displaying dissociation constants of 0.8 ꢁ 0.05
and 2 ꢁ 0.1 lM, respectively (Fig. 5B,D).
accommodate AF and SAM, and was located at the
interface between the Rossmann domain and the N-
terminal domain (Fig. 3A).
In addition, the web tools PROSITE [20], 3DLIGANDSITE
[21] and COACH [22] were used to predict and identify
ligand binding regions in RosA. PROSITE identified a
SAM binding region (residue 235–237) in the C-term-
inal Rossmann domain. This finding is in line with
results obtained using 3DLIGANDSITE, COACH and
CaSoX. 3DLIGANDSITE identified 25 crystal structures of
proteins structurally related to RosA with bound SAM
or SAH, and predicted 22 residues that are putatively
involved in cofactor binding to RosA. On the other
hand, the program COACH (http://zhanglab.ccmb.me-
d.umich.edu/COACH/) found several structures of
methyltransferases in complex with various ligands,
and clustered them according to a confidence score
(C-score between 0 and 1). The best-scored cluster pre-
Ternary complex formation with substrates and
products
The catalytic methylation of AF by RosA was investi-
gated by spectrophotometry using
a stopped-flow
device and conventional spectrophotometer,
a
4
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C. Tongsook et al.
Structure and mechanism of RosA
Fig. 1. Schematic representation of the
structure of RosA. (A) Cartoon
representation of the RosA protomer,
showing a-helices in red, b-sheets in
yellow, and loops in grey. (B) Cartoon
representation of the RosA dimer with one
protomer shown in red, yellow and grey,
and the other in pale blue, green and grey.
Fig. 2. Determination of the purity and
native molecular mass of RosA using SDS/
PAGE and FPLC. (A) Determination of the
subunit molecular mass of RosA after
purification by Ni-Sepharose FPLC was
performed by SDS/PAGE (12.5%). Lane 1,
low-molecular-mass protein marker;
lane 2, crude extract; lane 3, protein
fraction after purification by Ni-Sepharose
FPLC. The subunit molecular mass of
RosA was estimated to be 38 kDa. (B)
Determination of the native molecular
mass of RosA using Superdex 200 10/300
GL chromatography. (C) Plot of the
partition coefficient (Kav) against the
logarithm of molecular mass of standard
proteins (ferritin, 440 kDa; aldolase,
158 kDa; conalbumin, 75 kDa; ovalbumin,
43 kDa; ribonuclease A, 13.7 kDa). The
native molecular mass of RosA (filled
circle) was estimated to be 77 kDa.
exploiting the spectral changes that occur during
methylation
duct in both cases, suggesting that the observed spec-
tral changes are due to formation of a complex with
RosA and either SAM or SAH. The observed spectral
differences suggest considerable changes in the flavin
binding site upon formation of a ternary complex, and
the UV/Vis absorption spectra of RoF in various sol-
vents suggest that an apolar environment induces simi-
lar spectral changes to those observed in our
experiments (Fig. 6D). In addition, it is conceivable
that steric restriction induced in the ternary complex
[kmax (AF) = 478 nm;
kmax (RoF) =
505 nm]. At low concentrations of RosA, the final
absorption spectrum features an absorption maximum
at 505 nm, which is characteristic of complete forma-
tion of RoF (Fig. 6A). However, at high concentra-
tions of RosA the maximum at 505 nm is less
pronounced (hypsochromic effect), and additional max-
ima at a shorter wavelength are observed (Fig. 6B,C).
Analysis by HPLC revealed that RoF is the main pro-
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5

Structure and mechanism of RosA
C. Tongsook et al.
Fig. 3. Schematic representation of the
predicted binding pocket of RosA. (A)
Surface and cartoon representation of the
binding pocket of RosA predicted using
CASOX [19]. (B) Cartoon representation of
RosA with predicted binding of AF (shown
as yellow sticks) and SAM (shown as
orange sticks). Potential hydrogen bonding
interactions are indicated by blue dashed
lines. Water molecules are represented as
red spheres. The distance between the
reactive methyl group of SAM and the
amino group of AF is indicated by a green
dashed line.
Fig. 4. Structure of the putative active site of RosA. (A) Close-up view of the superimposed structures of RosA (shown in yellow) and
carminomycin-4-O-methyltransferase (PDB ID 1TW2; shown in blue). Ligands of 1TW2 (S-adenosylhomocysteine and 4-methoxy-E-
rhodomycin) are shown as blue sticks. AF and SAM are shown in their predicted binding modes as yellow and orange sticks, respectively.
(B) Close-up view of the predicted binding pocket of RosA. Potential hydrogen bonding with AF (shown in yellow) and SAM (shown in
orange) is indicated by blue dashed lines. Water molecules are represented as red spheres. The distance between the reactive methyl
group of SAM and the amino group of AF is indicated by a green dashed line.
prevents de-localization of the electron lone pair into
the aromatic system of the isoalloxazine ring, resulting
in the observed hypso- and hypochromic effect. There-
fore, we assume that formation of the ternary complex
generates a more apolar and sterically constrained envi-
ronment in the RoF binding pocket. In order to reveal
the nature of the ternary complex, we also determined
the affinity of RosA for SAM and SAH in the presence
of RoF. As shown in Fig. 7, the presence of RoF
greatly decreases the affinity for SAM, yielding a
dissociation constant of 180 ꢁ 20 lM, whereas the
affinity of SAH is approximately fourfold higher
(Kd = 0.4 ꢁ 0.05 lM). Thus the ternary complex with
SAH (product complex) is clearly favoured over a com-
plex with SAM (mixed substrate/product complex).
Next, we performed difference absorption spec-
troscopy to determine the dissociation constants of
RoF from the binary complex of RosA:SAH and
RosA:SAM, respectively, as well as the affinity of AF
for the RosA:SAH complex. The former experiments,
shown in Fig. 8A,B, yielded virtually identical dissoci-
ation constants of 14 ꢁ 2 and 15 ꢁ 2 lM, demonstrat-
ing that SAH and SAM cause similar decreases in the
binding affinity of RoF by a factor of 17–18. In con-
trast to product binding, AF binding to RosA is not
affected by SAH, as the determined dissociation con-
stant of 10 ꢁ 1 lM is almost identical to that observed
in the absence of SAH (Fig. 9). Table 3 lists the disso-
ciation constants obtained by ITC or spectrophoto-
metric titrations.
6
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C. Tongsook et al.
Structure and mechanism of RosA
Fig. 5. Binding of AF, RoF, SAM and SAH to RosA. Calorimetric titrations of RosA with AF (A), RoF (B), SAM (C) and SAH (D) were
performed in 50 mM Tris/HCl buffer, pH 8.0, containing 100 mM NaCl at 25 °C using a VP-ITC system (MicroCal). The dissociation constants
for binding of AF, RoF, SAM and SAH to RosA were 10 ꢁ 1, 0.8 ꢁ 0.05, 22 ꢁ 2 and 2 ꢁ 0.1 lM, respectively (three independent
measurements for each ligand). Data were analysed by non-linear least-squares fitting using ORIGIN version 7.0 (MicroCal).
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7

Structure and mechanism of RosA
C. Tongsook et al.
Fig. 6. UV/Vis absorption spectra at
various RosA concentrations. (A, B)
Spectral changes during the reaction of
5 lM AF and 0.8 mM SAM with 5 and
25 lM RosA, respectively. The reactions
were performed in 50 mM Tris/HCl buffer,
pH 8.0, containing 100 mM NaCl, in a
single-mixing stopped-flow
spectrophotometer. (C) A solution of
20 lM AF and 100 lM RosA was pre-
incubated prior to addition of 2 mM SAM.
All three reactions were run at 25 °C, and
absorption spectra were recorded from
350 to 600 nm using a diode array. (D)
Spectra for 25 lM RoF in various solvents
[water, dimethylsulfoxide (DMSO) and
ethyl acetate] and for 15 lM RoF in diethyl
ether.
Mechanistic studies of the dimethylation reaction
catalysed by RosA
that represent the consumption of the substrate
(k = 479 nm) and the generation of the product
(k = 530 nm). The reaction of RosA (20 lM) in the
presence of AF (20 lM) was investigated by mixing
with various concentrations of SAM (80–2000 lM).
The rates of the observed spectral changes at 479 and
530 nm were biphasic, and therefore fitted to two rate
equations (Fig. 11A). The first phase consisted of a
decrease in absorption at 479 nm and a concurrent
small increase at 530 nm (approximately 25%). This
first phase was interpreted to represent monomethyla-
tion of AF to MAF, and showed a hyperbolic depen-
dence on the SAM concentration, yielding a limiting
rate of 0.5 ꢁ 0.05 minꢀ1 and an equilibrium constant
of 180 ꢁ 40 lM (Fig. 11B). The second slower phase
corresponded to a decrease at 479 nm and a concomi-
tant increase at 530 nm. This phase showed an inverse
dependence on the SAM concentration approaching
0.05 ꢁ 0.002 minꢀ1 (Fig. 11C). The latter result sug-
gested that monomethylation of AF generates an inhi-
bitory product that prevents binding of SAM. In fact,
our binding studies have revealed that SAH binds
much more strongly to RosA than SAM does
(Table 3). Furthermore, the presence of RoF further
weakens SAM binding but the affinity for SAH is
increased (Table 3). Thus we reasoned that SAH,
which is produced in the first monomethylation reac-
tion, acts as a competitive inhibitor of SAM. To test
this hypothesis, we determined the rate of the methyla-
tion reaction as a function of SAM and SAH concen-
trations. The initial velocities were determined as a
With regard to the reaction mechanism, the structure
of RosA as well as the independent binding of AF and
SAM suggest that the enzyme operates by a random-
order kinetic mechanism, as shown in Scheme 2. To
test this hypothesis, we performed a series of pre-
steady-state kinetic measurements in the stopped-flow
device. In these experiments, different mixing orders
were employed, and the progress of the reaction was
monitored at 479 and 530 nm. As shown in Fig. 10A,
the rate of reaction was independent of the experimen-
tal set-up, i.e. whether RosA was directly mixed with
AF and SAM or pre-incubated with either of the sub-
strates. To obtain further insight into the kinetic mech-
anism, we performed a set of steady-state experiments
at various AF and RoF concentrations. As shown in
Fig. 10B, a primary reciprocal plot of the reaction
velocity against the reciprocal SAM concentration
resulted in a set of converging lines in accordance with
a random-order mechanism. From a re-plot of the pri-
mary data, i.e. slopes and y-axis intercepts versus the
reciprocal AF concentration (Fig. 10B, insets), the
Michaelis constants of AF and SAM were deduced to
be 4 and 70 lM, and the kcat was estimated as
0.06 minꢀ1 (Table 4).
As generation of RoF from AF requires two consec-
utive methylation steps, we also wished to obtain
insight into the individual methylation reactions.
Towards this aim, we analysed the reaction under sin-
gle-turnover conditions at two different wavelengths
8
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies

C. Tongsook et al.
Structure and mechanism of RosA
Fig. 8. Formation of a ternary complex comprising the RosA:SAH
or RosA:SAM complex with RoF. (A) A solution of RosA (25 lM)
and SAH (150 lM) was titrated with RoF (0–18.2 lM). The
absorption changes from the UV/Vis difference titrations were
recorded from 300 to 700 nm using a UV/Vis spectrophotometer.
The inset shows a plot of the absorption change at 510 nm as a
function of RoF concentration. A hyperbolic fit to the experimental
data yielded a dissociation constant of 14 ꢁ 2 lM. (B) A solution of
RosA (30 lM) and SAM (2 mM) was titrated in tandem cuvettes
with RoF (0–29.2 lM). The inset shows a plot of the absorption
Fig. 7. Formation of a ternary complex comprising the RosA:RoF
complex with SAH or SAM. (A) A solution of RosA (25 lM) and
RoF (5 lM) was titrated with SAH (0–3 lM). The absorption spectra
were recorded from 300 to 700 nm using
spectrophotometer. The inset shows plot of the absorption
change at 485 nm as function of SAH concentration.
hyperbolic fit to the experimental data yielded
a
UV/Vis
a
a
A
a
dissociation
constant of 0.4 ꢁ 0.05 lM. (B) A solution of RosA (25 lM) and RoF
(5 lM) was titrated in tandem cuvettes with SAM (0–990 lM). The
inset shows a plot of the absorption change at 485 nm as a
function of SAM concentration. A hyperbolic fit to the experimental
data yielded a dissociation constant of 180 ꢁ 20 lM. Both titrations
were performed in 50 mM Tris/HCl buffer, pH 8.0, containing
100 mM NaCl.
change at 510 nm as
a
function of the RoF concentration.
A
hyperbolic fit to the experimental data yielded
a
dissociation
constant of 15 ꢁ 2 lM. Both titrations were performed in 50 mM
Tris/HCl buffer, pH 8.0, containing 100 mM NaCl.
Fig. 12A, allows determination of the inhibition con-
stant of SAH as 7 lM. This value is in the same range
as the dissociation constant of SAH in the presence of
RoF, suggesting that MAF and RoF have similar
effects on SAH binding (Table 3). Thus we conclude
that the observed rate decline of the second kinetic
phase is due to the competitive effect of SAH, with the
limiting value corresponding to koff of SAH.
function of SAM concentration at various fixed con-
centrations of SAH to obtain information on the type
of inhibition. However, this plot did not allow us to
discern the inhibition type, i.e. competitive, non-com-
petitive or uncompetitive. As it was shown that SAH
binds more tightly to RosA than SAM does, we used
the linearized Henderson equation for data analysis
[23]. The resulting Henderson plot, as shown in
Fig. 12A, yielded a set of lines that converge on the
y axis. A re-plot of the slopes, as shown in the inset to
In a similar set of experiments, it was shown that
RoF is a competitive inhibitor of AF, exhibiting a Ki
value of 27 lM (Fig. 12B), which represents the disso-
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
9

Structure and mechanism of RosA
C. Tongsook et al.
Table 3. Dissociation constants (Kd, lM) of RosA for AF, RoF, SAH
and SAM, determined by ITC and UV/Vis absorption titrations.
Values marked with asterisk were obtained from ITC
measurements. Values marked with hash symbols (#) were
obtained from UV/Vis absorption titrations.
In the
presence of
AF
RoF
SAM
SAH
–
10 ꢁ 1*
0.8 ꢁ 0.05*
22 ꢁ 2*
2 ꢁ 0.1*
RoF
SAM
SAH
180 ꢁ 20#
0.4 ꢁ 0.05#
15 ꢁ 2#
14 ꢁ 2#
10 ꢁ 1#
RoF, i.e. the dissociation constant for SAM increased
approximately eightfold and that for SAH decreased
fourfold. These relative binding affinities clearly sug-
gest that the ternary complex observed in spectropho-
tometric experiments (Fig. 6) consisted of RoF and
SAH bound to RosA. The fact that the tightest binary
complex formed is that between RosA and RoF also
suggested a potential mechanism to control the con-
centration of free RoF in S. davawensis. It may be
assumed that the concentrations of both SAM and
SAH are too low to form a ternary complex with
RosA:RoF, and thus RoF inhibits binding of AF and
further generation of the product. This scenario is also
supported by the competitive inhibition of the RosA-
catalysed methylation of AF by RoF (Fig. 12). The
resistance of S. davawensis to RoF appears to be
related to the ability of the ribB FMN riboswitch to
discriminate between FMN and roseoflavin-5’-phos-
phate [7]. However, it is not clear yet whether other
additional mechanisms contribute to resistance in
S. davawensis. Therefore, it is plausible that the tight
binding of RoF to RosA constitutes an additional
mechanism to confer resistance towards RoF.
Although it is still unknown how RoF is eventually
secreted, it is conceivable that RosA also serves as a
chaperone to guide RoF to the secretion machinery
that is in charge of its translocation from the cell into
the environment.
Fig. 9. AF binds independently to RosA in the presence of SAH. A
solution of RosA (25 lM) and SAH (150 lM) was titrated with AF
(0–34.0 lM). The absorption spectra of the titrations were recorded
from 300 to 700 nm using a UV/Vis spectrophotometer. The inset
shows a plot of the absorption change at 495 nm as a function of
SAH concentration.
A hyperbolic fit to the experimental data
yielded a dissociation constant of 10 ꢁ 1 lM.
ciation constant of RoF in the presence of SAM (com-
pare Tables 3 and 5).
Discussion
Structure analysis revealed that RosA adopts a topol-
ogy that is characteristic of SAM-dependent methyl-
transferases. Although attempts to obtain structural
information for complexes with AF, SAM and SAH
were unsuccessful, further inspection of the active site
and the AF and SAM binding pockets provided useful
hints for mechanistic considerations that were tested
by binding studies and steady-state as well as pre-
steady-state kinetic experiments. The final biosynthesis
of RoF from AF by RosA is unusual in the sense that
the enzyme performs two consecutive methylations in
the same active site, and hence SAH must dissociate to
make room for another SAM while the monomethy-
lated intermediate, MAF, may remain bound to the
flavin-binding pocket. This complexity of the overall
conversion of AF to RoF was analysed by binding
studies as well as kinetic experiments to evaluate the
interaction of substrates and products in the RosA-cat-
alysed reaction. Much to our surprise, our binding
studies revealed that both products, RoF and SAH,
bind approximately ten times more tightly to RosA
than AF and SAM do (Table 3). Interestingly, the
affinity of RoF decreased approximately 20-fold in the
presence of SAM and SAH, whereas the affinities of
SAM and SAH respond differently to the presence of
The X-ray crystallographic structure of RosA sug-
gested that both substrates, AF and SAM, bind inde-
pendently to their respective pockets. This feature was
confirmed by kinetic experiments that demonstrated a
random-order binding mechanism for the first methy-
lation reaction yielding MAF and SAH (Fig. 10 and
Scheme 2). The rate of monomethylation of AF to
MAF showed a hyperbolic dependence on the SAM
concentration,
yielding
a
limiting
rate
of
0.5 ꢁ 0.05 minꢀ1 (Fig. 11B). Surprisingly, the subse-
quent reaction phase was characterized by an inverse
dependence on the SAM concentration, approaching a
10
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies

C. Tongsook et al.
Structure and mechanism of RosA
Scheme 2. Scheme of the methylation
reactions catalysed by RosA following a
random-order ‘bi-bi’ mechanism.
limiting rate of 0.05 ꢁ 0.002 minꢀ1 (Fig. 11C). As
noted above, SAH binds tightly to RosA, and its affin-
ity is enhanced in the presence of RoF (Table 3). It is
likely that the binding affinity of SAH in the presence
of MAF is in the same range, i.e. between 2 ꢁ 0.1 and
0.4 ꢁ 0.05 lM, and thus the second methylation reac-
tion depends on the rate of dissociation of SAH (koff).
This koff appears to be the limiting step for conversion
of MAF to RoF, and therefore the intrinsic rate for
this reaction step is not accessible in our kinetic experi-
ments. A summary of the reaction steps characterized
in this study is shown in Scheme 3.
formed as described previously [11]. E. coli Rosetta 2
(DE3) was transformed using pET24a(+)rosA-His6 (pFJ02)
[11]. One of the transformant strains was used to inoculate
a pre-culture that was aerobically incubated at 37 °C for
16 h (200 rpm) in lysogeny broth containing 50 lgꢂmLꢀ1
kanamycin. The cells were harvested by centrifugation
(5000 g at 4 °C for 30 min), and washed three times using
PBS (10 min per wash at 4 °C). The cells were then sus-
pended in 30 mL PBS and used as an inoculum for a 15-L
bioreactor (Bioengineering AG, Wald, Switzerland). Cell
growth was allowed to proceed for 7 h at 750 rpm, 37 °C,
with an aeration of 1 L of oxygen per L of culture per min-
ute in a total volume of 10 L until the cells reached an
absorption at 600 nm of 0.8. Synthesis of the recombinant
protein was induced by addition of 0.5 mM isopropyl-b-D-
thiogalactopyranoside. The culture was further incubated
for 16 h at 30 °C until an attenuance at 600 nm of 1.2 was
reached. Cells were harvested by centrifugation at 5000 g
(4 °C).
Experimental procedures
Reagents
All chemicals and reagents were of the highest purity commer-
cially available from Sigma-Aldrich (St. Louis, MO, USA)
and Merck (Darmstadt, Germany). AF was a generous gift
To produce selenomethionine-containing hexahistidine-
tagged RosA, the expression protocol for native RosA was
slightly altered. E. coli BL834 (DE3) was transformed with
pFJ02, and incubated for 7 h at 37 °C and 200 rpm in
selenomethionine minimal medium [26] supplemented with
50 lgꢂmLꢀ1 kanamycin. The pre-culture was harvested by
centrifugation at 5000 g (4 °C) and used as an inoculum
for a 15-L bioreactor (Bioengineering AG). Synthesis of the
recombinant protein was induced at when the cells reached
an attenuance at 600 nm of 0.8 by adding 0.5 mM
isopropyl-b-D-thiogalactopyranoside. Cells were harvested
when they reached an attenuance at 600 nm of 1.2, by cen-
trifugation at 5000 g (4 °C). All cell pellets were frozen
immediately after harvesting and stored at ꢀ80 °C.
€
from Sandro Ghisla (Universitat Konstanz, Germany). Ni-
NTA-agarose was obtained from GE Healthcare (Little Chal-
font, UK). RosA was over-produced in Escherichia coli, and
purified as previously described [11]. The concentrations of
the following compounds were determined spectrophotomet-
rically
usingꢀ1 these
extinction
coefficients:
ꢀ1
AF,
e490 = 42.0 mM ꢂcmꢀ1; RoF, e505 = 31.1 mM ꢂcmꢀ1 [24];
SAM and SAH, e260 = 15.4 mMꢀ1ꢂcmꢀ1 [25]; RosA (based on
amino acid sequence) = 27.1 mMꢀ1ꢂcmꢀ1
.
Expression and purification
TM
€
Heterologous production and purification of recombinant
The enzyme was purified using an AKTApurifier sys-
hexahistidine-tagged RosA from S. davawensis was per-
tem (GE Healthcare). Frozen cell pellets of E. coli
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
11

Structure and mechanism of RosA
C. Tongsook et al.
Table 4. Kinetic parameters for the RosA reaction.
Parameter
Value
KmAF (lM)
4
KmSAM (lM)
kcat (minꢀ1
70
)
0.06
20 mM imidazole, and was supplemented with protease
inhibitors (Roche cOmpleteTM EDTA-free protease inhibitor
cocktail, Roche, Basel, Switzerland). For purification of
SeMet-RosA, the HisTrap buffer also contained 2 mM b-
mercaptoethanol. The cells were disrupted using a French
press (three cycles, 2 9 108 Pa, 10 °C). The cell-free extract
was cleared by centrifugation at 10 000 g for 30 min at
4 °C. The supernatant was again centrifuged at 100 000 g
for 30 min at 4 °C to remove cell debris and unbroken
cells. The cleared lysate was filtered (0.45 lm), equilibrated
with loading buffer (20 mM Na2HPO4, pH 7.4, 500 mM
NaCl, 20 mM imidazole, plus 2 mM b-mercaptoethanol for
SeMET-RosA) and applied to a 5 mL HisTrap column
(HisTrapTM HP; GE Healthcare). Gradient elution of the
hexahistidine-tagged protein was performed using 0 to 50%
elution buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl,
500 mM imidazole, plus 2 mM b-mercaptoethanol for
SeMET-RosA). Eluted enzyme fractions were pooled and
desalted using a HiTrapTM desalting column (GE Health-
care), and equilibrated in a buffer containing 20 mM Tris/
HCl, pH 8.0, with 2 mM b-mercaptoethanol for SeMET-
RosA only. To achieve enzyme purity appropriate for
crystallization experiments, an additional ion exchange
chromatography step was performed. The enzyme fraction
was applied to a MonoQ 5/50 GL column (GE Healthcare)
and eluted using a linear gradient from 0 to 100% elution
buffer comprising 20 mM Tris/HCl, pH 8.0, 500 mM NaCl
(plus 2 mM b-mercaptoethanol for SeMET-RosA only).
Aliquots of the fractions were analysed by SDS/PAGE with
staining using Coomassie Brilliant Blue R-250. The homo-
geneous fractions were tested directly for RosA activity (see
below).
Fig. 10. Steady-state kinetics of RosA and substrate binding
sequence of the RosA reaction. (A). The RosA reaction was
performed at 25 °C in a stopped-flow spectrophotometer using
different mixing orders for the substrates AF and SAM. The traces
shown represent AF consumption and RoF formation, respectively,
at 479 nm (solid lines) and 530 nm (dotted lines). The red trace
represents the reaction of RosA:AF (5 lM RosA and 5 lM AF) with
80 lM SAM. The blue trace represents the reaction of RosA:SAM
(5 lM RosA and 80 lM SAM) with 5 lM AF. The green trace
represents the reaction of RosA (5 lM) with AF (5 lM) and SAM
(80 lM). All solutions were prepared in 50 mM Tris/HCl buffer, pH
8.0, containing 100 mM NaCl. (B) Double-reciprocal plot of the two-
substrate kinetics of the RosA reaction. The enzyme catalytic
assay was performed at 25 °C in a spectrophotometer, measuring
the increase in RoF formation at 530 nm. The reaction contained
5 lM RosA in 50 mM Tris/HCl buffer, pH 8.0, containing 100 mM
NaCl plus various concentrations of AF (20–100 lM) and SAM (20–
250 lM) as indicated. Insets 1 and 2 represent secondary plots of
the slope and y-axis intercept obtained from the primary plot
against reciprocal AF concentration, resulting in a KmAF of 4 lM, a
Determination of native and subunit molecular
masses of RosA
To determine the subunit molecular mass of RosA purified
protein was loaded onto SDS/PAGE (12.5%). Protein
molecular mass markers used were Low Molecular Weight
protein markers (GE Healthcare, Little Chalfont, UK) con-
taining phosphorylase b (97 kDa), albumin (66 kDa), oval-
bumin (45 kDa), carbonic anhydrase (30 kDa), trypsin
inhibitor (20 kDa), and a-lactalbumin (14.4 kDa). The gel
was stained with Coomassie Brilliant Blue R-250 prior to
destaining (10% ethanol, 10% glacial acetic acid in water).
To determine the native molecular mass of RosA gel fil-
tration chromatography at 25 °C was performed. The pro-
KmSAM of 70 lM, and a kcat of 0.06 minꢀ1
.
Rosetta 2 (DE3) and BL834 (DE3) over-producing hexahis-
tidine-tagged RosA and SeMet-RosA, respectively, was
resuspended in 50 mL HisTrap binding buffer. This buffer
contained 20 mM Na2HPO4, pH 7.4, 500 mM NaCl and
12
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies

C. Tongsook et al.
Structure and mechanism of RosA
tein solution was loaded onto a Superdex 200 10/300 GL
column attached to a AKTApurifierTM system (GE Health-
care) and then eluted with elution buffer (50 mM Tris-HCl,
for binding of RoF and AF to the RosA:SAH complex by
means of difference spectrophotometry, titrations were per-
formed at 25 °C in tandem cuvettes. All solutions were pre-
pared in 50 mM Tris/HCl buffer, pH 8.0, containing
pH 8.0 containing 100 mM NaCl) at
a flow rate of
0.5 mLꢂminꢀ1. Protein standard markers used in this exper-
iment were ferritin (440 kDa), aldolase (158 kDa), conalbu-
min (75 kDa), ovalbumin (43 kDa), and ribonuclease a
(13.7 kDa).
100 mM NaCl. A solution (0.8 mL) containing RosA
(25 lM) and SAH (150 lM) was placed in one of the two
chambers of the sample and reference cell. The other cham-
ber of each tandem cuvette was filled with the same volume
of buffer only. After recording a baseline, RoF (0–18.2 lM)
or AF (0–34.0 lM) were added to the solution containing
RosA and SAH in the sample cell and to the buffer in the
reference cell at 2 min intervals. Absorption spectra were
recorded from 300 to 700 nm after each titration step. Sim-
ilar titrations of the RosA:SAM complex (30 lM RosA and
2 mM SAM) with RoF were performed by addition of RoF
(0–29.2 lM) to the complex in the sample cell and to the
buffer in the reference cell. For determination of the disso-
ciation constants for SAH and SAM from the binary
RosA:RoF complex (25 lM RosA and 5 lM RoF), SAH
and SAM were titrated to final concentrations of 3 and
990 lM, respectively. Absorption changes were recorded
from 300 to 700 nm, and were plotted against the concen-
tration of ligands. The dissociation constants were obtained
by fitting the data using Levenberg–Marquardt algorithm
(Eqn 1) implemented in the KALEIDAGRAPH software (Syn-
ergy Software, Reading, PA, USA):
Isothermal titration calorimetry (ITC)
All of the experiments to determine dissociation constants
(Kd) for AF, RoF, SAM and SAH to RosA were per-
formed at 25 °C in 50 mM Tris/HCl buffer, pH 8.0, con-
taining 100 mM NaCl using a VP-ITC system (MicroCal,
Northampton, MA, USA). All solutions were degassed
before measurements. Titration experiments for AF and
RoF binding to RosA (30 lM, 1.42 mL) consisted of 20
injections (15 lL; duration, 30 s; spacing time, 250 s) of a
solution containing 0.6 mM AF and 0.48 mM RoF. Deter-
mination of the Kd for SAM and SAH was performed by
titration (20 injections) of 0.35 mM SAM (15 lL; duration,
30 s; spacing time, 250 s) and 0.48 mM SAH (15 lL; dura-
tion, 30 s; spacing time, 250 s) to a RosA solution (30 lM,
1.42 mL). The ITC raw data were analysed using ORIGIN
version 7.0 (MicroCal).
DAmax½Lꢃ
DA ¼
ð1Þ
Kd þ ½Lꢃ
Spectrophotometric titration of RosA with AF,
RoF, SAM and SAH
Steady-state kinetics of RosA
UV/Vis absorption spectra were recorded using a Specord
200 Plus spectrophotometer (Analytik Jena, Jena, Ger-
many) at 25 °C. To determine the dissociation constants
RosA catalyses the N,N-dimethylation of AF to yield RoF
as the final product. Steady-state kinetic measurements were
Fig. 11. Kinetic reaction of RosA:AF (1 : 1 ratio) with SAM. (A) Reaction of RosA:AF with SAM. A solution of RosA (20 lM) and AF (20 lM)
in 50 mM Tris/HCl, pH 8.0, containing 100 mM NaCl, was mixed with various concentrations of SAM (80–2000 lM), and the reaction was
monitored by the absorption change at 25 °C on a spectrophotometer. All concentrations are shown as final concentrations. The absorption
decrease at 479 nm (left y-axis) and increase at 530 nm (right y-axis) are shown. (B) kobs values for the first phase of the reaction
representing monomethylation step were plotted as a function of concentration of SAM, resulting a rate constant of 0.5 ꢁ 0.05 minꢀ1 with
a Kd of 180 ꢁ 40 lM. (C) Plot of kobs for the second phase against the concentration of SAM. The plot was fitted by non-linear curve fitting,
consistent with a rate constant of 0.05 ꢁ 0.002 minꢀ1
.
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
13

Structure and mechanism of RosA
C. Tongsook et al.
performed at 25 °C using a spectrophotometer. The assay
reaction contained 5 lM RosA, various concentrations of
AF (20–100 lM), and various concentrations of SAM (20–
250 lM) in 50 mM Tris/HCl buffer, pH 8.0, containing
100 mM NaCl. Progress of the RosA reactions was moni-
Pre-steady-state reaction studies
The methylation of AF bound to RosA was investigated at
various concentrations of SAM at 25 °C using a spec-
trophotometer. Briefly, a solution of RosA (20 lM) and AF
(20 lM) in 50 mM Tris/HCl buffer, pH 8.0, containing
100 mM NaCl was pre-incubated at 25 °C for 3 min, and
then the reaction was initiated by adding SAM (final con-
centrations of 80, 200, 400, 1000 and 2000 lM). Progress of
the reaction due to consumption of AF and formation of
the final product (RoF) was monitored at 479 and 530 nm,
respectively. Data analysis of the kinetic traces was per-
formed using the exponential equations from KINETIC STU-
DIO software (TgK Scientific Ltd) to obtain the observed
rates (kobs). Rate constants were determined from plots of
kobs as a function of the SAM concentration using the
Levenberg–Marquardt algorithm implemented in the KALEI-
DAGRAPH software (Synergy Software).
tored at 530 nm (e530 = 25.2 mM ꢂcmꢀ1), at which wave-
ꢀ1
length RoF formation may be monitored without
interference from other chromophores. Initial rates were
obtained using KINETIC STUDIO software (TgK Scientific Ltd,
Bradford-on-Avon, UK). A double-reciprocal plot of the
initial rate and the substrate concentration provides the
steady-state kinetic parameters, and also provides informa-
tion on the mechanism of a two-substrate enzyme reaction
according to Dalziel’s equation (Eqn 2), in which e/v is the
ratio between the enzyme concentration and the initial veloc-
ity, [A] and [B] are the concentrations of substrates A and B,
respectively, and Φ values are Dalziel’s coefficients [27]:
e
v
UA UB
UAB
¼ U0 þ
þ
þ
ð2Þ
½Aꢃ ½Bꢃ ½Aꢃ½Bꢃ
Stopped-flow spectrophotometric studies
All experiments were performed at 25 °C using an SF-
61SX2 stopped-flow spectrophotometer (TgK Scientific
Ltd, Bradford-on-Avon, UK) with a 1 cm path length.
Prior to all experiments, the stopped-flow instrument was
flushed with 50 mM Tris/HCl buffer, pH 8, containing
100 mM NaCl several times. To analyse the effect of sub-
strate binding order on the reaction kinetics, experiments
with different mixing orders of AF and SAM were per-
formed. Various pre-mixed solutions of RosA, RosA:AF or
RosA:SAM complexes were prepared, and then mixed with
solutions containing AF and SAM, SAM or AF, respec-
tively. The progress of the reactions was monitored at 479
and 530 nm using a KinetaScan diode array detector (TgK
Scientific Ltd).
Product inhibition of RosA
Enzymatic activity of RosA in the presence of the reaction
products RoF and SAH was studied by measuring the ini-
tial rate of the reaction at 25 °C using a spectrophotome-
ter. The assay reaction contained 5 lM RosA, various fixed
concentrations of AF (20–100 lM) and various fixed con-
centrations of SAM (20–100 lM) in 50 mM Tris/HCl buffer,
pH 8.0, containing 100 mM NaCl. Concentrations of RoF
in the range of 0–40 lM were used with various concentra-
tions of AF and a fixed concentration of SAM (250 lM).
The inhibition by SAH was analysed at various concentra-
tions of SAH and SAM using a fixed concentration of AF
(200 lM). Initial rates were obtained from linear data fitting
using KINETIC STUDIO software (TgK Scientific Ltd). A Hen-
derson plot of [I]/(1 – vi/v0) and the reciprocal of the frac-
tional initial velocity (v0/vi) (Eqns 3 and 4) was used to
obtain inhibition constants and information on the inhibi-
tion mechanism [23]:
Product analysis by HPLC
HPLC analysis of AF, MAF and RoF was performed
using an Atlantisꢀ dC18 reversed-phase column (5 lm,
4.6 9 250 mm; Waters, Milford, MA, USA) on an Ulta-
mate 3000 HPLC instrument (Dionex, Sunnyvale, CA,
USA) at 25 °C. All samples prepared from RosA reactions
were loaded onto the column and eluted using a multi-step
gradient of 100 mM formic acid and 100 mM ammonium
formate (pH 3.7)/methanol (0–3 min, 30% methanol; 3–
20 min, 30–75% methanol; 20–22 min, 75% methanol) at a
flow rate of 0.8 mLꢂminꢀ1. Detection of AF, MAF and
RoF was performed by measuring the absorption at 479,
490 and 509 nm, respectively.
ꢀ
ꢁ
½Iꢃ
v0
vi
¼ Kai pp
þ ½Eꢃ
ð3Þ
vi
v0
1 ꢀ
and for a competitive inhibitor:
ꢀ
ꢁ
½Sꢃ
Kai pp ¼ Ki 1 þ
ð4Þ
Km
In Eqns 3 and 4, [I] is the concentration of inhibitor (i.e.
RoF and SAH), v0 and vi are the initial velocity of a reac-
tion in the absence and presence of a inhibitor, respectively,
[E] is the total enzyme concentration, [S] is the variable
substrate concentration, Km is the Michaelis constant, Ki
represents the inhibition constant of a tight binding inhibi-
tor and Kai pp represents the apparent inhibition constant.
Crystallization
Crystallization drops containing purified native RosA at a
concentration of 20 mgꢂmLꢀ1 in 20 mM Tris/HCl, pH 8.0,
14
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies

C. Tongsook et al.
Structure and mechanism of RosA
Table 5. Product inhibition pattern and dissociation constants (Ki)
of the RosA–product inhibitor complex. Product inhibition was
fitted to the tight-binding inhibitor model.
Product
Type of inhibition
Ki (lM)
RoF
Competitive (AF)
27
7
SAH
Competitive (SAM)
Crystals of native RosA grew within 5 days after mixing
the protein solution (in a 1 : 1 ratio) with a solution con-
taining 200 mM sodium chloride, 100 mM Tris/HCl, pH
5.5, and 25% w/v PEG-3350.
The same approach was used to grow crystals of the
selenomethionine-labelled derivative. Equal amounts
(0.5 lL) of the variant at a concentration of 20 mgꢂmLꢀ1
in 20 mM Tris/HCl, pH 8.0, 500 mM NaCl and 2 mM b-
mercaptoethanol were mixed with precipitant containing
100 mM MES/imidazole, pH 6.5, 20 mM D-glucose, D-man-
nose, D-galactose, L-fructose, D-xylose and N-acetyl-D-glu-
cosamine, 10% w/v PEG-20000 and 20% v/v PEG-MME
550 [28]. SeMet-RosA crystals grew within 7 days after
crystallization set-up. For diffraction data collection, crys-
tals were harvested from their mother liquor using Cryo-
LoopsTM (Hampton Research, Aliso Viejo, CA, USA), and
flash-cooled in liquid nitrogen without any additional cry-
oprotection.
Structure determination and refinement
ꢀ
Diffraction data to a resolution of 2.2 A were collected for
native RosA from a single triclinic crystal (space group P1)
at beamline ID23-1 of the European Synchrotron Radia-
tion Facility (Grenoble, France). MAD data to a resolution
Fig. 12. Product inhibition of the RosA reaction. (A) SAH inhibition
of the RosA reaction. A Henderson plot for tight-binding inhibition
by SAH with respect to SAM was obtained by plotting [I]/(1 – vi/v0)
as a function of v0/vi at various fixed concentrations of SAM as
indicated. Initial rates of RosA reactions were measured by the
absorption change at 530 nm over time. The reactions contained
5 lM RosA, 200 lM AF, various fixed SAM concentrations (20–
100 lM), and various concentrations of SAH (0, 10, 20, 40 and
80 lM). The inset is a secondary plot of Kiapp, slopes obtained from
the primary plot, against the concentration of SAM yielding a Kiapp
of 7 lM. (B) RoF inhibition of the RosA reaction. A Henderson plot
for tight-binding inhibitor of product inhibition by RoF with respect
to AF was obtained by plotting [I]/(1 – vi/v0) as a function of v0/vi at
various fixed concentrations of AF as indicated. Initial rates of
RosA reactions were measured by the absorption change at
530 nm by time. The reactions consisted of 5 lM RosA, 250 lM
SAM, various fixed AF concentrations (20–100 lM), and various
concentrations of RoF (0, 10, 15, 25 and 40 lM). The inset is a
secondary plot of Kiapp, slopes obtained from the primary plot,
against concentration of AF, yielding a KiROF of 27 lM.
ꢀ
of approximately 3.5 A were collected at beamline X06DA-
PXIII of the Swiss Light Source at the Paul Scherrer Insti-
tute (Villigen, Switzerland). Three MAD datasets were
collected from a single tetragonal SeMet-RosA crystal
(space group I4122) at various wavelengths (peak, inflec-
tion, remote) that were determined from an X-ray fluores-
cence scan recorded around the selenium absorption edge.
Data processing and scaling for the native and SeMet
MAD datasets were performed using XDS [29], SCALA and
AIMLESS [30].
The calculated Matthews coefficient [30,31] of the MAD
datasets indicated a 99.9% probability for the presence of
one RosA molecule per asymmetric unit of the crystal. To
identify selenium sites, the two MAD pipelines AUTO-RICK-
SHAW [32,33] and PHENIX AutoSol [34,35] were used. The
combination of these programs yielded first phases and a
partial structure. These phases were further used as input
phases for the automated chain-tracing/building programs
PHENIX AUTOBUILD [36] and BUCCANEER [37] to extend the
model and to improve its quality. The combined model of
both rebuilding programs produced interpretable electron
densities, with preliminary values of R = 37% and
500 mM NaCl, were set up with commercially available
screening solutions using the microbatch method on an
Oryx-7 crystallization robot (Douglas Instruments Ltd,
Hungerford, UK) at 293 K. Drops were prepared by mix-
ing equal amounts (1.0 lL) of protein and precipitant.
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies
15

Structure and mechanism of RosA
C. Tongsook et al.
Scheme 3. Suggested reaction scheme for the reactions catalysed by RosA based on our binding studies and kinetic measurements.
Rfree = 49%. Rfree values were computed from a set of ran-
domly chosen reflections (5%), which were not used during
refinement [38]. Structure refinement was performed using
the refinement programs PHENIX REFINE [34] and REFMAC
[39]. Model rebuilding was performed using BUCCANEER
[37] and COOT [40] by alternate automated rebuilding and
real-space fitting against rA-weighted 2 Fo – Fc and
Fo – Fc electron density maps together with least-squares
optimization.
electron density map, and accepted or rejected on the basis
of geometry criteria as well as refined B-factors. In later
stages of the refinement, four TLS groups per protomer
were defined based on an analysis using the TLSMD web
server [42]. The final model was refined to R = 20% and
Rfree = 24%. Validation of the structure was performed
using MOLPROBITY [43], yielding a Ramachandran plot with
97.20% of the residues in favoured regions, 2.65% in
allowed regions and 0.15% in disallowed regions. Data
statistics and details of structure refinement are given in
Table 1 (MAD data) and Table 2 (native data).
The improved MAD model was used as an initial molec-
ular replacement template for PHASER [41] to determine the
structure of native RosA in the triclinic crystal form.
Molecular replacement resulted in six molecules in the
asymmetric unit. Structure refinement and model building
were performed using PHENIX REFINE [34] and COOT [40] by
real-space fitting against rA-weighted 2 Fo – Fc and
Fo – Fc electron density maps and least-squares optimiza-
tions. Water molecules were placed into the difference
Prediction of the SAM/SAH binding site
In order to locate putative binding sites for the substrate
AF and the cofactor SAM, the program CASOX [19]
together with the web tools PROSITE (sequence-based predic-
tion) [20], 3DLIGANDSITE (sequence- and structure-based
16
The FEBS Journal (2016) ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies

C. Tongsook et al.
Structure and mechanism of RosA
prediction) [21] and COACH (sequence- and structure-based
prediction) [22] were used. The results of the cavity analysis
using CASOX were compared with consensus ligand binding
residues predicted by PROSITE, 3DLIGANDSITE and COACH.
COACH additionally generated putative enzyme–ligand
model complexes and ranked them according to an intrinsic
C-score. This C-score represents a confidence score for the
prediction. C-scores range between 0 and 1, with a higher
score indicating a more reliable prediction. The highest-
rated complex, together with other related methyltrans-
ferase structures, were superimposed with RosA to identify
ligand binding modes. The modelled RosA–ligand complex
was further energy-minimized using the program YASARA
with the AMBER03 force field, employing the standard
optimization protocol [44]. Structure alignments were per-
formed using PYMOL (https://www.pymol.org/).
Escherichia coli operates at the transcriptional and
translational level and regulates riboflavin biosynthesis.
FEBS J 282, 3230–3242.
4 Pedrolli DB, Jankowitsch F, Schwarz J, Langer S,
Nakanishi S, Frei E & Mack M (2013) Riboflavin analogs
as antiinfectives: occurrence, mode of action, metabolism
and resistance. Curr Pharm Des 19, 2552–2560.
5 Pedrolli DB, Jankowitsch F, Schwarz J, Langer S,
Nakanishi S & Mack M (2014) Natural riboflavin
analogs. Methods Mol Biol 1146, 41–63.
6 Pedrolli DB & Mack M (2014) Bacterial flavin
mononucleotide riboswitches as targets for flavin
analogs. Methods Mol Biol 1103, 165–176.
7 Pedrolli DB, Matern A, Wang J, Ester M, Siedler K,
Breaker R & Mack M (2012) A highly specialized flavin
mononucleotide riboswitch responds differently to
similar ligands and confers roseoflavin resistance to
Streptomyces davawensis. Nucleic Acids Res 40, 8662–
8673.
Acknowledgements
8 Pedrolli DB, Nakanishi S, Barile M, Mansurova M,
Carmona EC, Lux A, Gartner W & Mack M (2011)
The antibiotics roseoflavin and 8-demethyl-8-amino-
riboflavin from Streptomyces davawensis are
metabolized by human flavokinase and human FAD
synthetase. Biochem Pharmacol 82, 1853–1859.
9 Juri N, Kubo Y, Kasai S, Otani S, Kusunose M &
Matsui K (1987) Formation of roseoflavin from 8-
amino- and 8-methylamino-8-demethyl-D-riboflavin. J
Biochem 101, 705–711.
This work was supported in part by the Austrian
€
Fonds zur Forderung der Wissenschaftlichen For-
schung through projects P22361 and W901 (DK
‘Molecular Enzymology’) to P.M. and K.G. We thank
the beamline staff at the European Synchrotron Radi-
ation Facility (Grenoble, France) and the Swiss Light
Source (Villigen, Switzerland) for their support during
diffraction data collection.
10 Matsui K, Juri N, Kubo Y & Kasai S (1979)
Formation of roseoflavin from guanine through
riboflavin. J Biochem 86, 167–175.
11 Jankowitsch F, Kuhm C, Kellner R, Kalinowski J,
Pelzer S, Macheroux P & Mack M (2011) A novel N,
N-8–amino-8–demethyl-d–riboflavin dimethyltransferase
(RosA) catalyzing the two terminal steps of roseoflavin
biosynthesis in Streptomyces davawensis. J Biol Chem
286, 38275–38285.
12 Jankowitsch F, Schwarz J, Ruckert C, Gust B,
Szczepanowski R, Blom J, Pelzer S, Kalinowski J &
Mack M (2012) Genome sequence of the bacterium
Streptomyces davawensis JCM 4913 and heterologous
production of the unique antibiotic roseoflavin. J
Bacteriol 194, 6818–6827.
13 Kelley LA & Sternberg MJ (2009) Protein structure
prediction on the web: a case study using the Phyre
server. Nat Protoc 4, 363–371.
14 Shi J, Blundell TL & Mizuguchi K (2001) FUGUE:
sequence–structure homology recognition using
environment-specific substitution tables and structure-
dependent gap penalties. J Mol Biol 310, 243–257.
15 Claude J-B, Suhre K, Notredame C, Claverie J-M &
Abergel C (2004) CaspR: a web server for automated
molecular replacement using homology modelling.
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Author contributions
M.M. and P.M. initiated the project; C.T., M.K.U.,
M.M., K.G. and P.M. designed the experiments and
analysed the data; C.T. and F.J. expressed and purified
RosA; M.K.U. and K.G. crystallized RosA and deter-
mined the crystal structure; C.T. performed analytical
and biochemical experiments, and determined dissocia-
tion constants as well as kinetic parameters; C.T.,
M.K.U., M.M., K.G. and P.M. wrote the manuscript.
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