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A Mycobacterium tuberculosis ligand-binding Mn/Feprotein reveals
a new cofactor in a remodeledR2-protein scaffoldCharlotta S.
Anderssona and Martin Högboma,b,1
aStockholm Center for Biomembrane Research, Department of
Biochemistry and Biophysics, Stockholm University, Arrhenius
Laboratories for NaturalSciences C4, SE-106 91 Stockholm, Sweden;
and bDepartment of Cell and Molecular Biology, Uppsala University,
Biomedical Center, Box 596,SE-751 24 Uppsala, Sweden
Edited by Harry B. Gray, California Institute of Technology,
Pasadena, CA, and approved February 23, 2009 (received for review
December 20, 2008)
Chlamydia trachomatis R2c is the prototype for a recently
discov-ered group of ribonucleotide reductase R2 proteins that use
aheterodinuclear Mn/Fe redox cofactor for radical generation
andstorage. Here, we show that the Mycobacterium
tuberculosisprotein Rv0233, an R2 homologue and a potential
virulence factor,contains the heterodinuclear
manganese/iron-carboxylate cofac-tor but displays a drastic
remodeling of the R2 protein scaffold intoa ligand-binding oxidase.
The first structural characterization ofthe heterodinuclear
cofactor shows that the site is highly specificfor manganese and
iron in their respective positions despite asymmetric arrangement
of coordinating residues. In this proteinscaffold, the Mn/Fe
cofactor supports potent 2-electron oxidationsas revealed by an
unprecedented tyrosine-valine crosslink in theactive site. This
wolf in sheep’s clothing defines a distinct func-tional group among
R2 homologues and may represent a structuraland functional
counterpart of the evolutionary ancestor of R2s andbacterial
multicomponent monooxygenases.
bioinorganic chemistry � diiron � manganese � monooxygenase �
R2c
D iiron-carboxylate proteins perform some of the most
chemi-cally challenging oxidations observed in nature. The 2
beststudied groups are the bacterial multicomponent
monooxygenases(BMMs) and the ribonucleotide reductase R2 proteins.
BMMs usethe diiron cofactor to perform a 2-electron oxidation, the
O2-dependent hydroxylation of hydrocarbons, including the
hydroxy-lation of methane to methanol performed by soluble
methanemonooxygenase (MMO) via a Fe(IV)-Fe(IV) intermediate
(1–3).BMMs have different and usually broad substrate
specificities,including alkanes, alkenes, and aromatic compounds.
For thisreason the proteins and the bacteria that produce them are
of greatinterest for industrial and environmental applications,
such asbioremediation of contaminated soil. All diiron hydrocarbon
hy-droxylases/monooxygenases are multisubunit complexes
requiringdifferent protein components for activity, complicating
their use inbiotechnological applications (4–6).
Ribonucleotide reductases (RNRs) are the only identified
en-zymes for de novo synthesis of all four deoxyribonucleotides.
TheR2 subunit of Class-I RNRs is a homodimeric
diiron-carboxylateprotein that performs a 1-electron oxidation.
Standard R2s gener-ate an essential stable tyrosyl radical (Y�) via
a Fe(III)-Fe(IV)intermediate (7–10).
Diiron-carboxylate proteins have very similar metal sites,
coor-dinated by 4 carboxylates and 2 histidines, and are believed
to sharea common evolutionary ancestor (6, 11). Much effort has
gone intodefining the structural and chemical determinants that
direct thesystems to perform 1- or 2-electron chemistry (4, 12,
13). A peculiarR2, lacking the radical harboring tyrosine, was
identified in theimportant human pathogen Chlamydia trachomatis
(14, 15). It wassuggested that the 1-electron oxidizing equivalent
was stored at themetal site, as opposed to as a tyrosyl radical,
and that this could bean adaptation to produce a radical site that
is less sensitive toscavenging by reactive nitrogen and oxygen
species produced by the
host’s immune response. In addition, a number of proteins,
previ-ously assigned as standard R2s, were assigned to this group
ofChlamydia R2-like proteins, denoted R2c. Recently it was
shownthat CtR2c possesses a manganese/iron redox cofactor and that
theone-electron oxidizing equivalent is stored as a
Mn(IV)-Fe(III)species that replaces the Fe(III)-Fe(III)-Y� cofactor
in standard R2s(16, 17). Interestingly, it was also shown that not
only is theMn(IV)-Fe(III) form stable to incubations with H2O2, but
thereduced forms are efficiently activated by H2O2 treatment
(18).
The Mycobacterium tuberculosis R2c-like protein, Rv0233, is 1
ofthe 10 most up-regulated proteins, about 7-fold, in the
virulentH37Rv M. tuberculosis strain compared with the avirulent
bacillusCalmette–Guérin (BCG) vaccine strain and is therefore a
possiblevirulence factor and drug target candidate (19). M.
tuberculosis isthe causative agent of tuberculosis (TB), one of the
worst globalkillers, with an estimated 1.7 million yearly deaths
and a third of theworld’s population infected. The bacterium boasts
one of nature’smost elaborate lipid metabolisms that is also key to
its virulence,inherent drug resistance and ability to multiply
within the macro-phage (20). There is a rapid development of multi
drug-resistantstrains (MDR-TB) and extensively drug resistant TB
strains (XDR-TB), resistant also to injectable second-line drugs,
are emerging andpose a severe threat to TB control worldwide.
Identification of newTB drugs and drug targets is imperative
(21).
ResultsR2c-Like Proteins Form 2 Subgroups. When the R2c proteins
werediscovered, there were only a handful of sequences available
for thegroup (15). Now this number is above 50, allowing
detailedsequence analysis. This reveals that R2c-like proteins
actually form2 groups in a phylogenetic tree, 1 group including
CtR2c and 1group including M. tuberculosis Rv0233 (Fig. 1).
Alignments showthat the Rv0233 group does not contain the conserved
C-terminaltyrosine, known to participate in radical transfer from
R2 to thecatalytic R1 subunit and shown to be essential for
activity in CtR2c(9, 22) (Fig. S1). Moreover, a number of organisms
with fullysequenced genomes that contain proteins from the Rv0233
groupdo not contain any other protein component of a Class I
RNRsystem. Together, this suggests that there may be functional
differ-ences between the groups, despite an overall sequence
similarityand conservation of active site and other key
residues.
Author contributions: M.H. designed research; C.S.A. and M.H.
performed research; C.S.A.and M.H. analyzed data; and M.H. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors
have been deposited in theProtein Data Bank, www.pdb.org (PDB ID
code 3EE4).
1To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/cgi/content/full/0812971106/DCSupplemental.
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Protein Production and RNR Assay. Rv0233 was produced as
anN-terminal His6-affinity-tagged protein by overexpression in E.
coli.M. tuberculosis H37Rv possesses 1 R1 homologue, nrdE, and
3genes that encode R2 protein homologues: nrdF1, nrdF2, andRv0233,
presently annotated as nrdB. We were unable to obtain anyRNR
activity above background from Rv0233 with M. tuberculosis R1(MtR1)
either with or without the addition of 1 equivalent each ofFe(II)
and Mn(II) per protein monomer while the MtR2, encoded bynrdF2, was
highly active with MtR1 in the established [3H]CDP assay asalso
shown previously (23). Activities obtained were (activity rel-ative
to MtR1�MtR2 in %, average � 1 SD); MtR1�MtR2: 100 �7.1,
MtR1�Rv0233: 0.55 � 0.12, MtR1�Rv0233�Fe(II)�Mn(II):0.50 � 0.083,
MtR1 only, 0.43 � 0.083.
Overall Structure. The protein was crystallized and the
structuresolved by SAD phasing to 1.9-Å resolution (Table 1).
Rv0233 isa homodimer with the same interaction surface and geometry
asR2s, producing the well-known heart-shaped dimer (Fig. 2A).The
chain could be traced from residue 2 through 290, and theoverall
structure is virtually identical to that of R2s. The core8-helix
bundle is conserved, and the main differences areobserved in
helices �D, �E, and the last 2 helices, �G and �H[nomenclature as
defined in (24)] (Fig. 2B). The largest back-bone structural
differences are observed in the N- and C-termini.The position of
the C-terminus is interesting in comparison toR2s. The R2 C-
terminus is known to interact with R1 and theC-terminal 30–40
residues are disordered in all R2 structuresdetermined to date. The
last modeled residues, however, align inspace within a radius of
only �5 Å, most likely relevant for theinteraction with R1. In
Rv0233, the C-terminal 24 residues are alsodisordered but the last
ordered residue is separated by �30 Åcompared with R2s. The
difference in location of the C-terminusfurther supports the
sequence and biochemical data showing thatthe protein is not an R2
component of a class I RNR.
Ligand Binding. A striking difference compared to R2 proteins is
thepresence of a bound ligand that coordinates directly to the
metal site(Fig. 3A). The ligand is modeled as myristic (C14) acid
because ofthe fit to the electron density and the complete lack of
H-bondinteractions between the ligand tail and the protein. The
ligand isbound in a large continuous cavity, similar to the one
observed in
BMMs with large substrates, for example, toluene
monooxygenase(4, 25), extending from the metal site toward the
protein surfacemade up of the loop linking helices �G and �H (Fig.
3B). The cavityis narrow and hydrophobic close to the metal site
and widens, onceit has passed between helices �B and �E of the
metal coordinating4-helix bundle, to produce a larger cavity with
more H-bondpossibilities. The narrow part of the cavity is
completely occupiedby the lipid ligand, while the wider part of the
cavity is also solvatedby ordered water molecules. In the present
structure, the cavity isclosed but conformational changes in the
loop linking helices �Gand �H or rotamer changes of R59, E244,
L248, or Y249 wouldopen the cavity to bulk solvent. The cavity is
produced withoutsignificant movement of the protein backbone
compared with R2s.This is achieved by numerous substitutions from
larger to smallersidechains in combination with architectural
differences in thesecond shell of cavity lining residues that allow
a number of firstshell side chains to position differently,
creating the cavity space.Based on the properties of the ligand
binding cavity and the boundligand it seems most likely that the
protein is involved in thebacterium’s lipid metabolism. Studies to
define the in vivo substrateare initiated but complicated by the
pathogenicity and very exten-sive lipid metabolism of M.
tuberculosis, including several poorlydescribed
mycobacterial-specific pathways.
Structure of the Heterodinuclear Metal Cofactor. Protein
producedin standard rich LB media contains significant amounts of
bothmanganese and iron, approximately 0.7 and 1.2
equivalents,respectively, as determined by ICP-SFMS. The Mn/Fe
ratio isalso reflected in the relative intensity of the K-level
X-rayemission lines from the crystal. Addition of 2 mM MnCl2 to
theexpression medium shifts this relation close to unity. Thus,
nomatter if the protein is produced at Mn:Fe ratios in the
expres-
Fig. 1. Phylogenetic tree of R2 homologues where the canonical
radicalharboring tyrosine is replaced by phenylalanine. The
locations of C. tracho-matis R2c and M. tuberculosis Rv0233 are
indicated.
Table 1. Crystallographic data and refinement statisticsfor
Rv0233
Rv0233 � � 0.934 Å
Data collection MosflmBeamline ID14–1Wavelength, Å 0.934Space
group P3221Cell parameters
Å 54.57; 54.57; 176.65° 90; 90; 120
Resolution, Å 40–1.90 (2.00–1.90)Completeness, % 99.9
(99.4)Redundancy 5.2 (4.4)Rsym, % 6.9 (36.2)I/�I 17.1
(3.5)Refinement statistics Refmac 5.4.0073Resolution, Å 40–1.9No.
of unique reflections 24918No. of reflections in test set
1268Rwork, % 14.9Rfree, % 17.7No. of atoms
Protein 2319Metal ions 2Ligand 16Solvent 266
RMSD from ideal values*Bond length, Å 0.022Bond angle, °
1.699
Ramachandran outliers, %† 1.5
*Ideal values from (37).†Calculated using a strict-boundary
Ramachandran plot (38).
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sion media of roughly 1:10 (rich media) or 100:1 (rich media �2
mM MnCl2), it still contains close to 1 equivalent of both Mnand Fe
per polypeptide.
The metal binding properties of the protein suggest to us that
thisprotein, like the related C. trachomatis R2, contains the
Mn/Fe-carboxylate redox cofactor. Anomalous diffraction difference
mapsshow that the metal binding is specific with the manganese
ionoccupying the site closest to the position of the radical
harboringtyrosine in standard R2s, thus replacing Fe1 (Fig. 4A).
The anom-alous data were collected on protein produced in
Mn-supple-mented media. Still, there is no Mn anomalous signal from
theFe-site above the noise level of the map. Similarly, there is no
signof Fe binding in the Mn site. The metal binding is thus very
specific,given that both metal ions are present in sufficient
amounts. Themanganese ion has fewer carboxylate coordinations,
despite thesymmetric arrangement of coordinating residues, this
differencemay affect metal specificity. However, the coordination
of theheterodinuclear cofactor is very similar to that observed in
diironBMMs and R2s. In these systems there is also known to be a
largeflexibility in metal coordination depending on oxidation state
andcoordinating exogenous ligands (3, 4, 26, 27). The basis for
thestrikingly strict metal specificity should lie in the metal free
andreduced M(II)-M(II) forms because it is at this oxidation state
thatthe metals bind to the protein. It seems very likely that the
metal
specificity in this system involves outer-sphere effects. The
presentstructure together with existing data on diiron systems
shouldprovide a useful tool to study the fundamental processes of
metalspecificity and redox tuning in proteins. The metal site
surroundingsand H-bonding distances are depicted in Fig. 4B and C.
Thenon-coordinating HxO species that is H-bonded to both the
exog-enous ligand and Y175 refines to a distance of 3.0 Å from
themanganese ion and does not seem to coordinate it directly;the
electron density for this ligand is also somewhat weaker than
forthe most well ordered water molecules, indicative of dynamics
orpartial occupancy.
Tyrosine-Valine Crosslink in the Active Site. The catalytic
potential ofthe metal site is manifested in the protein by the
formation of an
Fig. 2. Structure of Rv0233. (A) Overall dimeric structure of
the protein. (B)Superposition of Rv0233 (blue), E. coli R2 (green),
and C. trachomatis R2c (red).Helices �D, �E, �G, and �H display the
largest differences, the positions of theC-termini are
indicated.
Fig. 3. Ligand binding and cavity in Rv0233. (A) Ligand binding
and inter-action with the metal site shown by an Fo-Fc omit map for
the ligandcontoured at 0.42 e�3. (B) The ligand-binding cavity in
Rv0233 shown by theprotein molecular surface, the bound ligand is
displayed as VdW spheres,ordered water molecules in the cavity are
indicated. Residues R59, E244, L248,or Y249, restricting bulk
solvent access are shown as sticks.
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unprecedented tyrosine-valine crosslink, likely generated
duringone of the first redox cycles of the metal site. The phenolic
oxygenof Y162 is covalently bound to C� of V71, connecting the
metal sitecoordinating helices �B and �E (Fig. 5). The net chemical
reactionis a 2-electron oxidation of the V71-Y162 side chain pair
withremoval of 2 hydrogen atoms, resulting in the crosslink. With
theexception of a few reported cases of peptides containing
hydroxy-valine, this result is to our knowledge a previously
undocumentedmodification of a valine side chain in a protein.
Modification of thevery inert aliphatic side chain suggests that
the Mn/Fe cofactor iscapable of similarly challenging 2-electron
oxidations as BMMs.The use of the heterodinuclear site is thus not
limited to generatingand storing a one-electron oxidizing
equivalent as in CtR2c (16).Crosslinking of these amino acids,
which are also conserved in thegroup, possibly prepares the active
site for subsequent enzymaticchemistry (see Discussion).
We have considered if there could be alternative explanations
forthe formation of the crosslink than via oxidation by the
heterod-inuclear site. The only option in this case would be the
action ofanother enzyme. The crosslink is deeply buried and, to be
acces-sible, more than half of the protein would need to be
unfolded.Moreover, this would also imply that the expression host,
E. coli,possesses a system to form this crosslink in a M.
tuberculosis proteinthat has no close homologues in E. coli. This
possibility appearsextremely unlikely and cannot be considered a
real option forcrosslink formation.
A Combination of Conserved Features from both R2s and BMMs. In
thedirect vicinity of the metal site, the ligand-binding cavity is
estab-lished by the substitution of a phenylalanine residue, which
isabsolutely conserved in R2s, to an alanine (A171) conserved in
theRv0233 group (Fig. S1). This positions the cavity in the same
placeas the active site in BMMs. Moreover, in certain R2 mutants
thatare engineered toward 2-electron chemistry, this
phenylalaninebecomes hydroxylated (12, 28). In the present
structure, the boundligand occupies the same position in space and
is thus located at thepreferred location for substrate oxidation in
diiron carboxylateproteins.
The hydrogen bonding network on the histidine side of the
metalsite is known to control electron transfer and tuning between
1- and2-electron chemistry. In this area, the Rv0233 group displays
a
Fig. 4. Rv0233 active site. (A) Anomalous difference maps.
Purple: manga-nese anomalous difference density, contoured at 0.09
e�3. Green: iron-specific ddano map, contoured at 0.07 e�3. (B
and C) Metal-site coordinationand hydrogen bonding network of
conserved residues in the metal sitesurrounding, distances in
Å.
Fig. 5. Covalent crosslink between V71 and Y162 shown by an
Fo-Fc omitmap for the residues contoured at 0.42 e�3. The
�-helical part of �E isillustrated by the n � 5 main chain
H-bonds.
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composite structure of features otherwise unique to R2s or
BMMs(Fig. S1 and Fig. 4 B and C). The residues preceding both
metalcoordinating histidines are absolutely conserved as arginines
inBMMs, indicating that they are essential for function or folding
(4).The corresponding residues are mainly hydrophobic in R2s
andR2cs. The Rv0233 group, like the BMMs has conserved
positivelycharged residues in these positions, although the first
is a lysine(K103). This indicates that these residues in BMMs and
the Rv0233group are involved in electron transfer or redox tuning,
rather thannecessary for folding because the structurally virtually
identicalR2cs lack them. In addition, the Rv0233 group also
possesses thetryptophan, W32 (W48 E. coli R2 numbering), normally a
hallmarkof R2s and involved as a radical species in cofactor
assembly (29).In BMMs, the large side chain of the arginine
preceding the firstmetal coordinating histidine occupies the same
position in space asthe conserved tryptophan in R2s. In Rv0233, on
the other hand, thecorresponding K103 amine is involved in a
�-cation interaction withthe pyrrole ring of W32, most likely
tuning its chemical propertiesand redox potential. The hydrogen
bonding network is thus alsovery similar to the one in the
stearoyl-acyl carrier protein desatu-rases, another group of
2-electron, lipid-oxidizing, diiron carboxy-late proteins that
display a different dimer interaction geometrythan R2s and BMMs
(30). Y222 in CtR2c was recently shown tocontribute in mediating
the 1-electron reduction of the Mn(IV)-Fe(IV) state to produce the
active Mn(IV)-Fe(III) state. Mutationof this residue slows down the
external 1-electron reduction, thusstabilizing the Mn(IV)-Fe(IV)
intermediate (22). The correspond-ing residue is not conserved as
an electron-relay competent residuein the Rv0233 group, something
that may stabilize the Mn(IV)-Fe(IV) state and contribute to direct
the protein to 2-electronoxidations.
DiscussionRecently, a group of R2 proteins was discovered (15).
This groupuses a heterodinuclear Mn/Fe redox cofactor that, upon
reactionwith molecular oxygen, yields a Mn(IV)-Fe(III) oxidation
state thatis used in place of the diiron-tyrosyl radical system of
standard R2s(16). This solution, which actually seems less complex,
also appearsto be less sensitive to certain radical scavengers.
Here, we show thatthe use of the Mn/Fe-carboxylate cofactor is not
limited to the R2cproteins but is also present in a new group of
ligand-bindingMn/Fe-carboxylate proteins. On the sequence level
this group iseasily confused with the Mn/Fe-containing R2c proteins
but thepresent structure allows assignment of available sequences
to thedifferent groups. There are a number of sequence particulars
thatstrongly indicate that the proteins in the new group are all
ligand-binding oxidases, and we thus propose that they are
denoted‘‘R2-like ligand binding oxidases.’’ The in vivo substrates
andproducts are presently unknown, and may well differ within
thegroup. It seems most likely that the proteins are
hydrocarbonoxidases, possibly involving oxygen insertion. The
potential forchallenging 2-electron oxidations by the
heterodinuclear cofactor isdemonstrated by the formation of an
unprecedented tyrosine-valine crosslink in the active site. This
shows that the Mn/Fecofactor has a richer chemical repertoire than
the generation andstorage of a one-electron oxidizing equivalent,
as in CtR2c, and maybe similarly versatile as the
diiron-carboxylate cofactor.
We also describe the detailed structure of this cofactor.
Eventhough this structure is for a protein that is not an R2 the
extensivestructural similarities between the groups strongly
suggest that theR2c proteins also have the same arrangement with
the Mn ionsubstituting for Fe1. Since it is known that the
manganese assumesan (IV) oxidation state and serves as the radical
initiator in theactive state of CtR2c its position has great
importance for theradical transfer in these systems and should also
have implicationsfor the details of radical transfer in the
canonical diiron R2 proteins.
Because the residues involved in the tyrosine-valine crosslink
areconserved, it likely has relevance for protein function, this
role is not
obviously apparent. Some interesting features in relation to
BMMscan however be noted. Binding of the regulatory protein in
BMMsinduces structural changes in the active site that increase
oxygenreactivity and turnover as well as influence the
regiospecificity forhydroxylation (3). CD and MCD studies on MMO
show that thisis mainly a result of structural changes around Fe2,
in particular ofE209, corresponding to E167 in Rv0233 (31). In the
complexbetween phenol hydroxylase and its regulatory protein the �E
helixadopts a �-helical structure in this region. It was proposed
that thisfeature might mediate the effect of the regulatory protein
to theactive site (25). A recent study of toluene-4-monooxygenase
showsthat effector protein binding induces a number of structural
changesin the metal site coordinating helices, especially �E,
leading tochanges in both metal ligation and the active-site
channel (32). TheY-V crosslink in Rv0233 puts a strict geometric
restraint betweenhelices �B and �E and establishes a �-helix in �E,
comprising 2 fullturns and including E167, illustrated by the n � 5
main chainH-bonds in Fig. 5. The �E helices in BMMs are more
distorted interms of straightness than �E in Rv0233; still it is
noteworthy thatthe same segment adopts a �-helical structure. A
possible conse-quence is that the crosslink functions as a poor
man’s regulatoryprotein, imposing geometric restraints that fix the
helix in its�-conformation and thus lock the protein in one of
several statesotherwise controlled by the regulatory protein in
BMMs. It remainsto be shown whether this adduct has additional
functions or takespart in the chemistry as a cofactor.
Reconstitution of the Mn/Fe cofactor in CtR2c involves
aMn(IV)-Fe(IV) oxidation state (33). This is interesting because
theFe(IV)-Fe(IV) state has never been observed in an R2
proteinwhile methane monooxygenase is known to use this
intermediatefor substrate oxidation. The phenolic oxygen of the
cross-linkedY162 is located 5.1 Å from the iron ion. This is the
same distanceas the buried radical harboring tyrosine in canonical
R2s is to Fe1(ranging from � 4.6–6.6 Å depending on species and
oxidationstate). By analogy with these systems we hypothesize that
thecovalent link may be created via a mechanism involving a
Y162radical produced by the Mn(IV)-Fe(IV) oxidation state (Fig.
S2).
Interestingly, the phenolic oxygen of another conserved
tyrosine,Y175, present in all proteins in the Rv0233 group, but
conserved asPhe in R2cs and standard R2s, is H-bonded to the metal
site ligandE202 and via a water molecule to E68 (Fig. 4B and C).
The phenolicoxygen of Y175 lines the substrate-binding cavity and
is positioned4.9 Å from the manganese ion. Similarly to the radical
harboringtyrosine in R2s, Y175 is also linked by H-bonds to the
metal site.It thus seems reasonable that Y175 can become oxidized
to a radicalspecies by the metal site. However, unlike the R2s that
bury thetyrosyl radical in a hydrophobic pocket in the protein,
this residueis exposed to the substrate. In the present structure
Y175 is alsoH-bonded to the exogenous ligand via a water molecule.
This opensthe possibility that substrate oxidation and formation of
the cova-lent crosslink proceed via similar mechanisms involving
tyrosylradical-linked high valent metal site intermediates. Based
on thepositions of the HxO species, reactive metal-oxygen
intermediatesproduced by dioxygen cleavage are expected to reside
on thesubstrate-binding side of the metal site close to Y175,
providingpossibilities for oxygen insertion into the substrate.
From the pattern of sequence conservation it seems that
theprotein is a hybrid between BMMs and R2s. The conserved
featuresof the 2 diiron systems that are merged in Rv0233 must,
however,be interpreted in light of that the protein has a high
specificity forMn and Fe and that it is the heterodinuclear
cofactor that producesthe tyrosine-valine crosslink. The R2-like
ligand binding oxidasesdescribed here close the circle of a group
of proteins that performboth fascinating and important chemistry.
They merge structuraland functional features from 2 well-studied
families, the R2s andBMMs. This group of proteins should provide a
key tool toconsolidate and test theories about mechanistic
differences andsimilarities.
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Evolution of BMMs is believed to have ensued via a
geneduplication of a diiron carboxylate protein and subsequent
diver-gence into the catalytic �-subunit and the non-catalytic
�-subunit,while retaining the overall fold and dimer interaction
geometry.Accessory protein subunits were likely acquired via
horizontal genetransfer, which also largely characterizes the
spread of BMMs (6,11). The Mn/Fe-carboxylate proteins could
represent structural andfunctional homologues of an ancient
ancestor of R2s and BMMsand Mn/Fe could be considered a possible
candidate for theancestral cofactor. Although the evolutionary
relationship is clearlythe topic for more detailed analysis, it is
apparent that bothfunctions can be housed in very similar
homodimeric proteinscaffolds with a heterodinuclear
Mn/Fe-carboxylate cofactor.
Materials and MethodsDetailed materials and methods are
described in SI Materials and Methods.
Bioinformatics. Sequences encoding R2 homologues but lacking the
canonicalradical harboring tyrosine were collected by sequence
database searching.
Cloning, Protein Expression, Purification, Enzymatic Assays, and
Metal Analysis.The Rv0233 gene was PCR cloned from M. tuberculosis
strain H37Rv (20) andoverexpressed in E. coli BL21(DE3) grown in LB
medium either without metalsupplement or with the addition of 2 mM
MnCl2. Protein was purified byaffinity and size exclusion
chromatography. Ribonucleotide reductase enzy-matic activity for M.
tuberculosis R1 with Rv0233 was measured using theestablished
[3H]CDP assay. Initial indication that the protein contained
more
than one metal was obtained by a simple combined luminescence
and color-imetric assay (34). Quantitative metal analysis was
performed using induc-tively coupled plasma sector field mass
spectrometry. The intensity of theX-ray K-level emission lines were
also used to estimate the relative amount ofMn and Fe in the
crystal as well as to verify that the crystallization process
didnot impose any significant enrichment of protein containing a
particularmetal.
Crystallization, Data Collection, and Structure Determination.
Rv0233 wascrystallized using the vapor diffusion method.
Diffraction data were collectedat the ESRF synchrotron in Grenoble,
France. Data collection statistics areshown in Tables 1, S1, and
S2. The intrinsic metal cofactor of the protein wasused to phase
the data by means of single-wavelength anomalous dispersionmethods
using anomalous data collected at the high energy side of the
ironedge. Model statistics are presented in Table 1. To determine
metal identity inthe different binding sites, anomalous diffraction
data were collected at thehigh-energy side of the Mn-edge (� � 1.8
Å) and the high-energy side of theFe-edge (� � 1.7 Å) (Table S2).
Anomalous difference model phased Fourier(DANO) maps were
calculated with FFT (35). At � � 1.8 Å, manganese, but notiron,
displays an anomalous signal and these data were used to determine
theposition of the Mn ion. Since both manganese and iron display
anomaloussignals at � � 1.7 Å, an iron-specific ‘‘difference DANO’’
map was calculatedusing both datasets according to (36).
Coordinates and structure factors aredeposited in the PDB with id
3EE4.
ACKNOWLEDGMENTS. We are very grateful to T. Alwyn Jones for
support anddiscussions. This work was supported by grants from the
Swedish ResearchCouncil and the Swedish Foundation for Strategic
Research to M.H. and T.A.J.and the Knut and Alice Wallenberg
Foundation to M.H.
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