Reversal of the substrate specificity of CMP N-glycosidase to dCMP

Article abstract of DOI:10.1021/bi400316p

MilB is a CMP hydrolase involved in the early steps of biosynthesis of the antifungal compound mildiomycin. An enzyme from the bacimethrin biosynthetic pathway, BcmB, is closely related to MilB in both sequence and function. These two enzymes belong to the nucleoside 2′-deoxyribosyltransferase (NDT) superfamily. NDTs catalyze N-glycosidic bond cleavage of 2′- deoxynucleosides via a covalent 2-deoxyribosyl-enzyme intermediate. Conservation of key active site residues suggests that members of the NDT superfamily share a common mechanism; however, the enzymes differ in their substrate preferences. Substrates vary in the type of nucleobase, the presence or absence of a 2′-hydroxyl group, and the presence or absence of a 5′-phosphate group. We have determined the structures of MilB and BcmB and compared them to previously determined structures of NDT superfamily members. The comparisons reveal how these enzymes differentiate between ribosyl and deoxyribosyl nucleotides or nucleosides and among different nucleobases. The 1.6 A structure of the MilB-CMP complex reveals an active site feature that is not obvious from comparisons of sequence alone. MilB and BcmB that prefer substrates containing 2′-ribosyl groups have a phenylalanine positioned in the active site, whereas NDT family members with a preference for 2′-deoxyribosyl groups have a tyrosine residue. Further studies show that the phenylalanine is critical for the specificity of MilB and BcmB toward CMP, and mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to dCMP.

Full text of DOI:10.1021/bi400316p

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Article
Reversal of Substrate Specificity of CMP N-glycosidase to dCMP
Megan D Sikowitz, Lisa E. Cooper, Tadhg P. Begley, Pierre Alexandre Kaminski, and Steven E Ealick
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi400316p • Publication Date (Web): 09 May 2013
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Reversal of Substrate Specificity of CMP N-glycosidase to dCMP†,‡
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Megan D. Sikowitz§, Lisa Cooper∥, Tadhg P. Begley∥, Pierre Alexandre Kaminski⊥, and
Steven E. Ealick§,*
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§Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853,
∥Department of Chemistry, Texas A&M University, College Station, Texas 77843, and
, ⊥Institut Pasteur, Unité de Chimie et Biocatalyse, UMR CNRS 3523, 75724 Paris Cedex
15, France.
† This work was supported by NIH grants GM73220 and T32GM008500, and by the
Robert A. Welch Foundation (Aꢀ0034 to TPB). This work is based upon research
conducted at the Advanced Photon Source on the Northeastern Collaborative Access
Team beamlines, which are supported by award GM103403 from the National Institutes
of Health. Use of the Advanced Photon Source is supported by the U.S. Department of
Energy, Office of Basic Energy Sciences, under Contract No. DEꢀAC02ꢀ06CH11357.
‡ The coordinates of MilB/SO4, MilB/CMP, and, BcmB/PO4 have been deposited in the
Protein Data Bank under accession codes 4JEN, 4JEM, 4JEL, respectively.
*To whom correspondence should be addressed at the Department of Chemistry and
Chemical Biology, Cornell University, Ithaca, NY 14853. Telephone: (607) 255ꢀ7961.
Fax: (607) 255ꢀ1227. Eꢀmail: see3@cornell.edu
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ABBREVIATIONS
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NDT, nucleoside 2′ꢀdeoxyribosyltransferase; hmCMP, 5ꢀhydroxymethyl cytidine
5′ꢀmonophosphate; CMP, cytidine 5′ꢀmonophosphate; SeMet, selenomethionyl; AMP,
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adenosine 5′ꢀmonophosphate; IMP, inosine 5′ꢀmonophosphate; UMP, uridine 5′ꢀ
monophosphate; dCMP, 2′ꢀdeoxycytidine 5′ꢀmonophosphate; GMP, guanosine 5′ꢀ
monophosphate; SAD, singleꢀwavelength anomalous diffraction; PTD, purine
deoxyribosyltransferase; DMHA, N,Nꢀdimethylhexylamine; 5MD, 5ꢀmethylꢀ2'ꢀ
deoxypseudouridine;
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ABSTRACT
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MilB is a CMP hydrolase involved in the early steps of biosynthesis of the
antifungal compound mildiomycin. An enzyme from the bacimethrin biosynthetic
pathway, BcmB, is closely related in both sequence and function to MilB. These two
enzymes belong to the nucleoside 2′ꢀdeoxyribosyltransferase (NDT) superfamily. NDTs
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catalyze Nꢀglycosidic bond cleavage of 2′ꢀdeoxynucleosides via a covalent 2ꢀ
deoxyribosylꢀenzyme intermediate. Conservation of key active site residues suggests that
members of the NDT superfamily share a common mechanism; however, the enzymes
differ in their substrate preferences. Substrates vary in the type of nucleobase, the
presence or absence of a 2′ꢀhydroxyl group, and the presence or absence of a 5′ꢀ
phosphate group. We have determined the structures of MilB and BcmB and compared
them to previously determined structures of NDT superfamily members. The
comparisons reveal how these enzymes differentiate between ribosyl and deoxyribosyl
nucleotides or nucleosides, and among different nucleobases. The 1.6 Å structure of the
MilBꢀCMP complex reveals an active site feature that is not obvious from sequence
comparisons alone. MilB and BcmB that prefer substrates containing 2′ꢀribosyl groups
have a phenylalanine positioned in the active site whereas NDT family members with
preference for 2′ꢀdeoxyribosyl groups have a tyrosine residue. Further studies show that
the phenylalanine is critical for MilB and BcmB specificity towards CMP, and mutation
of this phenylalanine residue to tyrosine results in a 1000ꢀfold reversal of substrate
specificity from CMP to dCMP.
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MilB is an enzyme involved in the biosynthesis of the natural product mildiomycin
(Figure 1A), a commercially available antifungal agent isolated from Streptomyces
rimofaciens.1ꢀ3 MilB preferentially hydrolizes the Nꢀglycosidic bond of 5ꢀhydroxymethyl
cytidine 5′ꢀmonophosphate (hmCMP) to release 5ꢀhydroxymethyl cytosine, which is
later incorporated into mildiomycin (Figure 1B).1 Previous studies found that MilB also
hydrolyzes cytidine 5′ꢀmonophosphate (CMP) to its cytosine and ribose 5′ꢀphosphate
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(Figure 1B).1 MilB is a member of the 2′ꢀdeoxynucleoside ribosyltransferase (NDT)
superfamily. Typically, NDTs catalyze the reversible transfer of 2′ꢀdeoxyribose from a
donor 2′ꢀdeoxynucleoside to an acceptor base. Their structural and functional properties
have been investigated in detail.4ꢀ6 NDTs are found in a limited number of organisms and
are thought to be important for nucleoside recycling when alternative nucleoside salvage
pathways do not exist.5 In contrast to most NDTs, MilB participates in a hydrolysis
reaction, in which the acceptor base is replace by a water molecule. While NDT exhibits
hydrolysis activity in the absence of an acceptor base, the hydrolysis activity is about
100ꢀfold lower than its transferase reaction.6
BLAST analyses starting with the MilB sequence suggest homology to BcmB
from Clostridium botulinum (47% sequence identity). Like MilB, BcmB also shows
CMP hydrolase activity, but is utilized in a different biosynthetic pathway. BcmB is
responsible for the production of the 5ꢀhydroxymethylcytosine precursor for the
biosynthesis of bacimethrin, a thiamin antimetabolite found in C. botulinum (Figure 1)
(T.P. Begley, manuscript in preparation). While MilB and BcmB both belong to the
NDT superfamily, they differ from other members of this family in their substrate
preferences. Most enzymes containing the NDTꢀmotif are specific for 2′ꢀ
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deoxynucleosides.4, 7 MilB and BcmB hydrolize hmCMP, a ribonucleotide containing 5′ꢀ
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monophosphate and 2′ꢀhydroxy groups.
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In addition to MilB and BcmB, several other enzymes containing the NDT motif
function as nucleoside transferases. BlsM from Streptomyces griseochromogenes (52%
sequence identity to MilB) is a nucleotide hydrolase, with CMP as the preferred
substrate. The cytosine produced from the BlsM reaction is incorporated into blasticidin
S, a commercially available antibiotic compound (Figure 1).8 Rcl, another member of
the NDT superfamily, catalyzes the hydrolysis of purine 2′ꢀdeoxyribosylnucleotides and
is inhibited by ribonucleotides 9. Its solution structure10, 11, and recent crystal structures
with bound inhibitors12, provide highly detailed information about its substrateꢀenzyme
interactions. Rcl is an unusual because it is the only NDT family member found in
mammals. Rcl is upregulated in some cancers and contributes to tumorigenesis;
however, the details of its cellular function are unclear.13, 14
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Because members of the NDT superfamily are not widely distributed and they
show significant variation in substrate specificity, their evolution is not clearly
understood. Conservation of key active site residues suggests a common catalytic
mechanism even though the substrates for different family members vary in their
preference for ribosyl or 2′ꢀdeoxyribosyl groups, 5′ꢀhydroxyl or 5′ꢀphosphate groups, and
the type of nucleobase. Scheme 1 shows the reaction for NDT with a deoxyribosyl
covalent intermediate, where Base1 and Base2 may be any purine or pyrimidine base.
Each half reaction proceeds with inversion of stereochemistry at C1′, thus, the full
reaction proceeds with retention of stereochemistry.
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Here we report the Xꢀray structures of MilB and BcmB, and of MilB complexed
with CMP. Comparisons with the structures of NDT6 and Rcl10ꢀ12 revealed a structural
basis, which was not obvious from sequence comparisons alone, by which NDT family
members differentiate between ribosyl and 2′ꢀdeoxyribosyl substrates. The selectivity is
attributed to a phenylalanine residue found in MilB and BcmB that is replaced by
tyrosine in NDT and Rcl. In support of this hypothesis, in vitro enzymatic activity assays
showed that for both MilB and BcmB, mutation of the active site phenylalanine residue
to tyrosine resulted in a reversal of substrate preference from CMP to dCMP.
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MATERIALS AND METHODS
Cloning, Expression, and Purification of MilB and BcmB. All cloning procedures
followed standard DNA manipulation methods. The S. rimofaciens ZJU5119 milB gene
was commercially synthesized by DNA2.0 and bcmB was cloned using genomic DNA
from C. botulinum A str. ATCC 19397
. The milB and bcmB genes were inserted into
pTHT, a modified pETꢀ28 plasmid (Novagen) containing an Nꢀterminal His6ꢀtag and
TEV protease recognition site. All mutant plasmids were prepared at the Cornell Protein
Production and Purification Facility by siteꢀdirected mutagenesis of the native gene
following standard, PCRꢀbased mutagenesis.15
Escherichia coli B834(DE3), a methionine auxotrophic cell line, was transformed
with the pTHTꢀmilB wildꢀtype (WT) plasmid. E. coli BL21(DE3) cells were used to
express all other proteins. Starter cultures were grown from a single colony in 10 mL of
sterile LB media containing 30 ꢁg/mL kanamycin (LBꢀkan) at 37 °C overnight with
shaking. Selenomethionine (SeMet)ꢀcontaining MilB was prepared in 1.5 Lꢀ cultures in
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minimal media with 0.4% glucose, 1x MEM vitamin mix, 2 mM MgSO4, 0.1 mM CaCl2,
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25 mg/L FeSO4, 20 mg/L all amino acids except methionine, and 50 mg/L of Lꢀ
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selenomethionine. To remove any LB media during SeMet protein preparation the
overnight culture was centrifuged at 1000g for 20 min before the cell pellet was collected
and resuspended in minimal media. The 1.5 L volume was then inoculated with 5 mL of
starter culture and allowed to grow at 37 °C with shaking until reaching an OD600 of 0.6,
at which point the incubator temperature was reduced. Once reaching an induction
temperature of 15 °C and OD600 ~0.8, protein expression was initiated by inoculating the
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cultures with 0.5 mM isopropyl βꢀDꢀthiogalactopyranoside. After 18 h, cells were
centrifuged at 2000g for 20 min, collected, and frozen at ꢀ20 °C for storage. For native
protein overexpression, 5 mL of overnight culture was added directly to 1.5 L of LBꢀkan
media. The cells were then grown, collected and stored as described for the preparation
of SeMetꢀMilB protein.
After thawing and resuspending the cell pellet in 45 mL of lysis buffer (50 mM
Tris, 300 mM NaCl, and 20 mM imidazole at a pH of 8.0), the cells were lysed on ice
with two rounds of sonication. The cell extract was centrifuged for 30 min at 40,000g at
4 °C after sonication. The supernatant was then collected and loaded onto a 2 mL Niꢀ
NTA column (Qiagen) preequilibrated with the lysis buffer. The column was washed
with 50 mL of lysis buffer to remove any nonspecifically bound contaminants. To elute
the protein, the column was washed with elution buffer (50 mM TRIS, 300 mM NaCl,
and 250 mM imidazole at a final pH of 8.0). The elution sample was collected and
further purified using size exclusion chromatography (HiLoad Superdex 200 PG; GE
Healthcare). The purification procedures for WT and mutant BcmB, WT and mutant
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MilB, and SeMet MilB all followed this protocol. The native MilbWT polyhistidineꢀtag
was removed for crystallization by incubation with TEV protease for 12 h at 4 °C. The
sample was passed over a NiꢀNTA column preequilibrated with lysis buffer and the flowꢀ
through volume containing MilB was collected. The purity of the sample was analyzed
by SDSꢀPAGE before concentration to ~20 mg/mL. Protein was buffer exchanged by
overnight dialysis into storage buffer (SeMet MilB: 50 mM Tris, pH 8.5, 500 mM NaCl;
WT MilB: 10 mM Tris, pH 8.0, 100 mM NaCl; BcmB: 10 mM Tris pH 8, 50 mM NaCl)
before flash freezing with liquid nitrogen for storage at ꢀ80 °C.
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Crystallization of MilB and BcmB. The hangingꢀdrop vapor diffusion method was
used for crystallization of both MilB and BcmB. After mixing equal volumes of protein
sample and reservoir solution, the samples were equilibrated overnight at 18 °C against a
total reservoir volume of 500 ꢀL. Protein concentrations for crystallization were 10
mg/mL for MilB and 15 mg/mL for BcmB. Initial crystallization conditions were
determined using commercially available sparse matrix screens (Hampton Research,
Emerald Biosystems).
SeMetꢀMilB crystals grew as hexagonal rods approximately 200 ꢀm long and 40
ꢀm wide under the optimized crystallization condition of 8% PEG 1000, 0.1 M
phosphateꢀcitrate pH 4.2, and 0.2 M Li2SO4 after two days. A second condition for MilB
crystallization was found with the Nꢀterminal polyhistidine tag removed. Square rods
400 ꢀm long by 20 ꢀm wide crystallized after two days in 0.1 M Tris pH 7.5, 0.2 M
MgCl2, and 18% PEG 8000. The ligand soaked into the crystals added using a solution
containing the components of the reservoir solution supplemented with 10 mM cytosine
and 10 mM ribose 5ꢀphosphate for 5 min before cryoprotection.
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BcmB crystals formed 200 ꢀm by 150 ꢀm plates with 10 ꢀm thickness in 18%
PEG MME 2000, 0.1 M phosphateꢀcitrate pH 4.4, 0.01 M spermine, and 0.2 M
(NH4)2SO4 after five days. All crystals were cryoprotected with a solution consisting of
their crystallization condition supplemented with 20% ethylene glycol before flash
freezing with liquid nitrogen.
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Data Collection and Processing. Xꢀray diffraction experiments were conducted
at the Advanced Photon Source NEꢀCAT beamline 24ꢀIDꢀC (Argonne National Lab)
using a Quantum315 detector (Area Detector Systems Corp.) using vitrified crystals. A
fluorescence scan was conducted to determine the data collection wavelength
corresponding to the maximum f′′ for the SeMetꢀMilB crystals. All data collection runs
used the oscillation method with 1.0° rotation per frame. HKL2000 was used for
indexing, integrating and scaling the data.16 Data collection and processing statistics are
listed in Table 1.
Structure Determination, Model Building, and Refinement. The crystal structure
of MilB was solved by singleꢀwavelength anomalous diffraction (SAD) using SeMetꢀ
MilB. The crystals belong to space group P6222 with a Matthews coefficient of 2.1
Å3/Da and an estimated solvent content of 41%, assuming one protein chain per
asymmetric unit.17 Initial phase calculation and model building were carried out using
PHENIX AutoSol.18, 19 Subsequent model improvement was performed using COOT for
manual manipulations and PHENIX.refine with default parameters for structure
refinement.19, 20 Water molecules were added during later rounds of refinement in
PHENIX and the final structure was analyzed using COOT and MolProbity.21 The
structure of MilB complexed with CMP was determined in space group C2 with two
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protein chains per asymmetric unit. Under this new condition, MilB also crystallized
with a Matthews coefficient of 2.1 Å3/Da corresponding to 41% solvent content.
SKETCHER was used for ligand generation before placement into Fo − Fc electron
density.22 Model improvement was performed as described above.
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BcmB crystallized in space group C2 with three chains per asymmetric unit and a
Matthews number of 3.2 Å3/Da, corresponding to a solvent content of approximately
61%. The structure of BcmB was determined using molecular replacement, with the
newly solved MilB structure as the search model.23 MolRep placed three chains in the
asymmetric unit and further model building and refinement was carried out using COOT
and Refmac.20, 24 The three chains correspond to one dimer formed through
noncrystallographic twofold symmetry and one dimer generated by crystallographic
symmetry. Several rounds of refinement using Refmac and PHENIX.refine resulted in a
BcmB model with Rꢀfactor and Rfree values of 21.7% and 24.2%, respectively.
Refinement statistics for SeMetꢀMilB, MilBꢀCMP and BcmB are summarized in Table 1.
Analytical HPLC. Reaction mixtures were analyzed by reverseꢀphase HPLC on an
Agilent 1200 HPLC system equipped with a quaternary pump and thermostatted
autosampler (4 °C). The stationary phase was a Supelcosil LCꢀ18ꢀT column (15 cm × 4.6
mm, 3 ꢁm particles). The LC eluent (1 mL/min flow rate) consisted of a gradient of
methanol and 6 mM N,Nꢀdimethylhexylamine (DMHA) in water that was adjusted to pH
6.5 by addition of acetic acid. In the analytical method the percentages of DMHA (D)
and methanol (M) balanced with water at time t varied according to the following
scheme: (t,M,D): (0,0,100), (2,10,80), (12,20,60), (14,65,10), (16,0,100), (21,0,100).
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Chromatograms were detected using the absorbance at 270 nm. UVꢀVis spectra of
substrate and product peaks were also collected and assessed.
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Substrate Specificity for MilB and BcmB. Substrate specificity studies for MilB
and MilB F17Y were carried out with adenosine 5′ꢀmonophosphate (AMP), CMP, 2′ꢀ
deoxycytidine 5′ꢀmonophosphate (dCMP), guanosine 5′ꢀmonophosphate (GMP), and
inosine 5′ꢀmonophosphate (IMP) (Figure S1). Experiments were designed based on the
previously described assay conditions used for MilA/MilB 25. In vitro enzymatic assays
were prepared with a final volume of 50 ꢁL. AMP, CMP, dCMP, GMP, or IMP (1 mM)
were added to MilB WT or MilB F17Y (10 ꢁM) in 50 mM Tris buffer, pH 7.5 and then
the samples were incubated at 37 °C for 3 h. Control samples omitting enzyme were also
prepared. Reactions were quenched by boiling at 100 °C for 5 min. Samples were
diluted with DMHA, filtered using a 3 kDa MWCO membrane, and analyzed by HPLC
as described above. Reaction products were compared to commercially available
standards that were analyzed under identical conditions. Substrate specificity studies for
BcmB and BcmB F6Y were carried out with CMP and dCMP as described above.
Kinetics for MilB and BcmB. Kinetic parameters were monitored based on
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cytosine production from CMP or dCMP for MilB WT, MilB F17Y, BcmB WT, and
BcmB F6Y. Each enzyme was incubated with varying substrate concentrations (see Table
S1) in 50 mM Tris, pH 7.5 for 30 min at 37 °C and then the reactions were quenched by
boiling at 100 °C for 5 min. Samples were diluted with DMHA, filtered using a 3 kDa
MWCO membrane, and analyzed by HPLC as described above. Reaction products were
compared to commercially available standards that were analyzed under identical
conditions. The product absorbance signal was measured by integration (ChemStation,
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Agilent Technologies) and compared to cytosine standards of known concentration.
Kinetic parameters were calculated using the MichaelisꢀMenten function in
KaleidaGraph 4.0 (Synergy Software).
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Figure Preparation. All figures were prepared using PyMOL 26, ChemDraw
(Cambridge Biosoft), KaleidaGraph 4.0 (Synergy Software), or Excel (Microsoft), and
compiled in Photoshop (Adobe).
RESULTS
Structure of MilB. The MilB with bound SO4 contains one protomer per
asymmetric unit; however, a dimer is formed by crystallographic twofold symmetry. The
final model at 1.95 Å resolution contains residues 11ꢀ162 of the possible 170 amino acid
residues. The protomer of MilB contains an α/β fold with a fiveꢀstranded parallel βꢀsheet
with a strand order of 21345 (flavodoxinꢀlike). The βꢀsheet is flanked on both sides by
αꢀhelices in an asymmetric manner (Figure 2A and 2B). Dimer formation occurs through
interactions between helices α4, α5, and α6 of the two protomers with an interface area
of 1170 Å2. Size exclusion chromatography confirmed the dimeric state of MilB (data
not shown), which is consistent with the quaternary structures of both NDT (trimer of
dimers) and Rcl.
The MilB/CMP complex also crystallizes with one dimer per asymmetric unit.
The final model at 1.55 Å resolution contains residues 12ꢀ168 of chain A and residues
10ꢀ162 of chain B. Each protomer is occupied by one CMP molecule and the overall
structure is essentially the same as that of the MilB/SO4 complex. Each dimer features
two equivalent active sites.
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MilB Active Site. The MilB active site is located at the Cꢀterminal edge of the βꢀ
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sheet, between helices α4 and α5 (Figure 2C). All of the interactions necessary for
substrate binding and catalysis are contained within a single MilB protomer, with no
direct participation from the adjacent protomer. A phosphate/sulfate binding pocket
contains hydrogen bonding interactions with the Ser97 sidechain, the amide groups of
Gly99, Arg23, and Phe22, and two well ordered water molecules (Figure 2D). This
binding site corresponds to the phosphate binding site in the CMP complex. Ordered
water molecules occupy the rest of the active site in the MilB/SO4 structure.
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The structure of the MilB/CMP complex provides the molecular details of the
nucleobase and ribosyl binding sites (Figure 3). The products of the CMP hydrolysis
reaction, cytosine and ribose 5ꢀphosphate, were soaked into the MilB crystals.
Continuous electron density is observed in the active site representing CMP, the product
of the reverse reaction, yet our in vitro studies did not show significant formation of CMP
upon incubation of MilB, cytosine, and ribose 5ꢀphosphate (data not shown). Thus, the
presence of CMP in the MilB/CMP active site is a result of enzyme activity despite the
fact that we could not detect activity in vitro. Glu62 accepts a hydrogen bond from the 4ꢀ
amino group of the cytosine base and, if protonated, would donate a hydrogen bond to
CMP N5. Glu103 accepts hydrogen bonds from the CMP 2′ꢀ and 3′ꢀhydroxyl groups.
Additionally, the side chain of Asp78 is within hydrogen bonding distance of the cytosine
O2. Phe17 C4 is positioned 3.5 Å from the CMP 2′ꢀoxygen in the MilB structure.
Structure of BcmB. BcmB crystallized in space group C2, with three protein
chains in the asymmetric unit. The structure of BcmB was determined to 3.0 Å
resolution using the MilB structure (47% sequence identity) described above as the
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molecular replacement search model. The protomeric structure of BcmB displays the
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same α/βꢀtwist fold as MilB, with five parallel βꢀstrands forming a parallel βꢀsheet
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flanked on either side by αꢀhelices (Figures S2A and S2B). The final BcmB model
contains 120 amino acids in chain A, 123 in chain B, and 110 in chain C. The missing
regions include residues 11ꢀ20 and 110ꢀ113 in chain A, 11ꢀ21 and 112 in B, and 10ꢀ24,
and 109ꢀ112, which correspond to loop regions. Missing residues 47ꢀ55, 48ꢀ56, and 46ꢀ
58 in protomers A, B and C, respectively, correspond to a short αꢀhelix and loop when
aligned with MilB.
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Chains A and B form a dimer by twofold noncrystallographic symmetry. The
buried surface area at the interface is 1320 Å2. Chain C forms a dimer with an equivalent
chain C using crystallographic twofold symmetry. The buried surface area of this
interaction is 1210 Å2. This small discrepancy between the A/B and C/C interface
surfaces areas can be attributed to additional disordered regions in chain C. Residues 58
and 59 in A and B and residue 114 in chain A are present at the A/B dimer interface, but
are disordered at the C/C dimer interface. The oligomeric state of BcmB observed in the
crystal structure is supported by size exclusion chromatography results (data not shown)
and closely resembles the dimer formed by MilB (Figure S2C).
BcmB Active Site. The active site of BcmB is similar to that of MilB. Each active
site contains a bound phosphate ion from the crystallization solution. The phosphate
makes hydrogen bonds with the side chain of Ser86 and the amide backbone of Gly88 in
the phosphateꢀbinding pocket (Figure S2D). Based on structural comparison to MilB, the
BcmB active site residues that participate in substrate binding include Asp67, Glu92, and
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Phe6 (Figure 4). Glu41 falls in a disordered region in the BcmB crystal structure, but is
expected to be equivalent to Glu62 in MilB on the basis of sequence alignments.
Kinetics and Substrate Specificity for MilB and BcmB. Initial studies determined
the substrate specificity of MilB for cytosine containing nucleotides.1 MilB preferentially
hydrolyzed CMP, but cytosine formation was also observed, to a lesser extent, from
dCMP (Figure 5). MilB showed little to no hydrolysis of purine containing substrates
(Figure S1). CMP or dCMP was incubated with MilB F17Y and the production of
cytosine was monitored to measure Nꢀglycosidase activity. MilB F17Y successfully
cleaved dCMP but was no longer able to effectively hydrolyze CMP (Figure 5). Similar
studies were conducted for both BcmB wild type (WT) and the corresponding active site
Phe mutant (F6Y). As with MilB WT, BcmB WT can hydrolyze CMP and, to a lesser
extent, dCMP (Table 2). Conversely, BcmB F6Y hydrolyzed dCMP with a 2′ꢀ
deoxyribosyl group, while showing only minor activity with CMP (Figure S3).
Steadyꢀstate kinetic parameters were determined for MilB WT, MilB F17Y,
BcmB WT, and BcmB F6Y using either CMP or dCMP as substrates (Table 2). The
kcat/Km value for MilB WT with hmCMP was previously reported as 22.1 Mꢀ1sꢀ1.1 The
kcat/Km values observed for MilB WT hydrolysis of CMP or dCMP were of 5.2 Mꢀ1sꢀ1 and
1.1 Mꢀ1sꢀ1, respectively.1
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DISCUSSION
Structure Comparison. MilB and BcmB both adopt an α/βꢀfold composed of five
parallel βꢀstrands forming a core βꢀsheet flanked on either side by several αꢀhelices.
This fold is highly conserved among the NDT superfamily enzymes. A pairwise DALI
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comparison between the MilB and BcmB structures results in a Zꢀscore of 16.9 and
RMSD of 1.7, confirming their highly conserved tertiary structure (Table 3).27 A DALI
structure search against the PDB starting with MilB reveals structural homology to the
known 2′ꢀdeoxyribosyltransferases and Rcl (Table 3). Though their sequence identities
are relatively low (11ꢀ22%), the enzymes retain similar folds. Based on a Zꢀscore of 15.5
with a RMSD value of 2.3Å, MilB has the highest structural similarity to R. norvegicus
Rcl (PDB ID 4FYI)12. Similar RMSD values of 2.5 Å and 2.9 Å are observed upon
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comparison of MilB to the Rcl solution structures with PDB IDs 2KHZ11 and 2KLH10, 11
,
respectively. Several 2′ꢀdeoxyribosyltransferases, such as the Trypanosoma brucei30 and
L. leichmannii 6, 28, 29 NDTs (PDB ID 2F2T and 1F8X) and L. helveticus purine
nucleoside 2′ꢀdeoxyribosyltransferase (PTD) (PDB ID 1S2L)29 are also among the most
homologous structures.
While the enzymes in the NDT superfamily maintain a similar tertiary structure,
their oligomeric assembly is not fully conserved. Both NDTs and PTD form a hexamer
composed of a trimer of homodimers, while Rcl, MilB, and BcmB are observed to form
only dimers.6, 11, 28 Comparison of the dimer interfaces reveals a conserved mode of
interaction (Figure 6). Like many members of the NDT superfamily10, the interactions
observed at the dimer interfaces in MilB and BcmB are mainly hydrophobic (>97%).
Base Specificity. Structural analysis of MilB, BcmB, and related enzymes reveals
the basis of their substrate specificities. MilB shows specificity for the pyrimidine
containing hmCMP or CMP substrates. It does not hydrolyze the purine containing
nucleotides, demonstrated by an inability to hydrolyze AMP, GMP, or IMP (Figure S1).
While highly specific for hmCMP or CMP, its relatives display variable nucleobase
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specificity (Table 4). NDT accepts all naturally occurring and some synthetic
nucleosides, while PTD30 and Rcl7 are specific for purine containing nucleosides or
nucleotides, respectively. In the structure of MilB/CMP, the Glu62 carboxylic group is
positioned approximately 3 Å from the cytosine, forming hydrogen bonds with the
cytosine N3 and 4ꢀamine group (Figure 3). Helix α3 (on which Glu62 is located), helix
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α4, and the loop region formed by residues 67ꢀ70 shield the MilB active site. As a result,
MilB has a relatively compact substrateꢀbinding site that cannot accommodate the bulkier
purine containing nucleotides. BcmB specificity is predicted to function similarly to
MilB based on sequence and structural homology.
The carboxylic acid from the NDT Cꢀterminal Tyr157′ and Gln46 amine interact
with its substrate nucleobase. NDT can accommodate a variety of nucleobases because
of its flexibility in both accepting and donating hydrogen bonds.6 Like NDT, PTD
interacts with nucleobases via hydrogen bonds from the carboxylic tail of the Cꢀterminal
tyrosine located in the active site. PTD diverges from NDT, as there are no equivalent
interactions to those provided by Gln46 in NDT.28 The complexed Rcl structures 10ꢀ12
reveal a large nucleobase binding area with no specific enzymeꢀnucleobase interactions,
allowing Rcl to accommodate a variety of nucleobases in its active site. Because the Rcl
active site is rather open, it is solvent accessible and favors the larger purine containing
substrates that can aid in shielding the active site from the solvent. As evidenced by the
structures of MilB and other NDT superfamily members, their nucleobase specificities
are derived from a combination of favorable enzymeꢀsubstrate hydrogen bonding
interactions and active site accessibility.
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Specificty at the 5
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ꢀposition. The NDT family members differ in their substrate
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preference for either a hydroxl or phosphate group at the 5′ꢀposition (Table 4). The highꢀ
resolution MilB/CMP structure reveals key information about the enzymeꢀsubstrate
binding interactions at the CMP 5′ꢀposition, including seven hydrogen bonds. Two
ordered water molecules each contribute one hydrogen bond, while Ser97 is hydrogen
bonding distance from either of the two 5′ꢀphosphate oxygen atoms. While the active site
serine residue observed in MilB is conserved in both BcmB and BlsM (Figure 7), the
remaining interactions in the MilB phosphateꢀbinding pocket come from amide backbone
groups and ordered water molecules. The amide backbones of Phe22, Arg23, and Gly99
act as hydrogen bond donors, completing the phosphateꢀbinding pocket (Figure 8A).
Although the identities of the backbone residues are not conserved in sequence, the
phosphate ion in BcmB/PO4 is observed in an analogous binding pocket to MilB (Figure
8B). This pocket is also rich with hydrogen bond donors resulting is preference for
phosphate groups at the 5′ꢀposition.
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A 5′ꢀphosphate binding pocket utilized for nucleotide binding was also observed
in Rcl.10ꢀ12 The structure of Rcl shows hydrogen bonding contacts between the GMP
phosphate group and Ser17, Arg19, Ser87, and Ser117′ (Figure 8B). This motif is highly
conserved in the sequences of 13 different species in Rcl.10 While MilB does not share
this motif, the positively charged pocket is consistent with preference for nucleotide
monophosphate binding. In NDTs and PTD, an aspartate residue plus either an
asparagine or glutamine residue stabilizes nucleoside 5′ꢀhydroxyl groups, favoring
nucleoside binding (Figure 8C)6, 28 and preventing the binding of 5′ꢀphosphate
containing nucleotides preferred by MilB and BcmB.4
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Specificity at the 2
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ꢀposition. While MilB and BcmB contain the conserved NDT
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motif, their ability to hydrolyze the Nꢀglycosidic bonds of ribosylnucleotides makes them
unique from other enzymes in this class. NDTs, PTD, and Rcl show no glycosidase
activity towards ribosylꢀcontaining substrates, which in some cases are inhibitory.9
Conversely, MilB and BcmB show higher activity towards substrates containing 2′ꢀ
hydroxy groups (Table 2). In the related Rcl and NDT structures, a tyrosine residue is
observed near the substrate 2′ꢀbinding position (Figure 8). Not obvious from sequence
analysis alone, the structures of MilB and BcmB reveal a phenylalanine in the position
equivalent to this tyrosine near the conserved catalytic glutamate (Figure 7). Compared
to Rcl or NDTs, this phenylalanine residue is the only residue near the 2′ꢀbinding position
that differs in either MilB or BcmB.
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The identity of the active site phenylalanine or tyrosine residue proves to be
important in differentiating between ribosyl or deoxyribosyl containing substrates by
MilB and BcmB. Structural studies of PTD28, which has a tyrosine residue near the 2′ꢀ
binding position, suggested that ribosylated substrates form hydrogen bonds with the
catalytic glutamate, rendering it unreactive. This rational contradicts our findings in
which MilB and BcmB react with ribosylated substrates, despite also showing
interactions between their catalytic glutamate and the substrate 2′ꢀhydroxy group. When
both a hydroxy containing substrate and active site tyrosine are present, the glutamate
may be too constricted by hydrogen bonds to position itself for catalysis. In the absence
of an active site tyrosine, the catalytic glutamate is not restricted by the ribosylated
substrate. Rather, the ribosylated substrate may help orient the glutamate for catalysis.
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Acceptor/Nucleophile Binding Site. Previous studies of NDTꢀsuperfamily
members have explored the structural basis of nucleophile binding at the active site. In
NDT, the interaction between Gln46 and the nucleobase positions the loop region, on
which Gln46 is located, shielding the active site from solvent.28 Along with a
hydrophobic binding pocket, this excludes water as a nucleophile. After initial Nꢀ
glycosidic bond cleavage and release of the base, NDT binds the incoming acceptor base,
positioning it for the second half of the NDT transferase reaction. Due to more limited
substrateꢀenzyme interactions in PTD, the corresponding loop region does not shield its
active site28 and bulkier purine bases that shield the active site are favored over smaller
pyrimidines, both as substrate and acceptor nucleobases. Compared to NDT, Rcl has a
longer loop region that does not shield the active site from solvent. It also does not have
any residues that specifically interact with and bind acceptor nucleobases.10ꢀ12 These
structural features allow a water molecule to act as the final acceptor. Because the MilB
active site is shielded by αꢀhelices, it is less solvent accessible as compared to PTD or
Rcl. Compared to NDT, the MilB active site is similarly shielded, but is not as
hydrophobic. As a result, an active site water molecule is available to act as the final
acceptor. Accordingly, the MilB/CMP structure reveals a water molecule in the active
site positioned 4.9 Å from the 1′ꢀribosyl position.
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Mechanism of Hydrolysis. Previous studies showed that NDT family members
catalyze transferase reactions utilizing a covalent enzymeꢀ(deoxy)ribosyl intermediate.6, 9,
31
In the first half reaction the covalent intermediate forms between a glutamate side
chain and C1′ of the (deoxy)ribosyl group (Scheme 1). The second half reaction takes
place after the purine/pyrimidine base is released and a nucleophile (either a different
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base or a water molecule) displaces the glutamate side chain. Each half reaction is
inverting at C1′ such that the stereochemistry at C1′ is retained for the full reaction. The
catalytic glutamate residue is absolutely conserved among the NDTs, Rcl, MilB, BcmB,
and BlsM (Figure 8). Based on this mechanism, Glu103 of MilB forms a covalent
attachment to CMP at the ribosyl C1′ꢀposition in the first half of the hydrolysis reaction
(Scheme 2). Previous studies on xylanases, which utilize a glutmate/glycoside covalent
intermediate in a hydrolysis reaction similar to that of MilB, showed that each half
reaction proceeds through an oxocarbeniumꢀlike transition state.32 In the crystal structure
of the MilB/CMP complex the glutamate oxygen atom is not correctly oriented for a
strictly SN2 mechanism suggesting the formation of an oxocarbenium ion intermediate
followed by trapping of the intermediate by Glu103. The second half reaction, in which
water displaces the glutamate side chain, likely proceeds through an oxocarbeniumꢀlike
transition state. In MilB, Asp78 is positioned to protonate the cytosine base leaving
group and to activate a water molecule for the second half of the reaction. This aspartate
residue is conserved in MilB, BcmB, BlsM, and Rcl, also suggesting a conserved
mechanism of hydrolysis.
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Reversal of Substrate Specificity. Based on the structural data, we hypothesized
that the active site phenylalanine is the key residue that confers specificity for either
2′ꢀhydroxyl or 2′ꢀdeoxy groups. In MilB, Phe17 C4 is 3.6 Å from the catalytic glutamate
residue and 3.5 Å from the CMP 2′ꢀhydroxyl group. A tyrosine is at a position
equivalent to MilB Phe17 in all previously solved structures of enzymes in the NDT
superfamily, which prefer 2′ꢀdeoxy substrates (Figure 9). MilB F17Y was constructed
using siteꢀdirected mutagenesis and used to test if the phenylalanine to tyrosine change is
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sufficient to increase the efficiency of dCMP hydrolysis. While MilB WT preferentially
hydrolyzes CMP, with a fiveꢀfold lower efficiency towards dCMP, mutation of F17Y
MilB resulted a 100ꢀfold decrease in the hydrolysis of CMP and a 100ꢀfold increase in
the hydrolysis of dCMP, corresponding to a >10,000ꢀfold inversion of substrate
specificity (Table 2). A similar, but slightly smaller affect in substrate specificity was
observed for BcmB.
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The largest change is seen in the preference of F17Y MilB for dCMP compared to
CMP. The value of kcat for WT MilB with CMP is about 3ꢀfold larger than for that of
dCMP. For F17Y MilB, the value of kcat with dCMP is about 600ꢀfold larger than for
CMP. For both WT and F17Y MilB, the KM values for both substrates are in a similar
range with the value for F17Y MilB with dCMP being the lowest. The value of kcat for
WT BcmB with CMP is about 20ꢀfold larger than for that of dCMP. For F6Y BcmB, the
kcat with dCMP is about 20ꢀfold larger than for CMP. The KM values for WT and mutant
BcmB show a larger range compared to those of MilB. The lowest value is 0.042 mM
for F6Y BcmB with dCMP and the highest value is 0.9 mM for WT BcmB with dCMP.
In the structure of MilB/CMP, the CMP 2′ꢀhydroxyl group interacts with the
catalytic Glu103, orienting the molecule in the active site. Based on the location of the
Phe17 in MilB/CMP, the MilB F17Y Tyr17 hydroxyl group would be 3.1 Å from the 2′ꢀ
hydroxyl group of bound CMP. This may cause unfavorable interactions for CMP
binding or cause electronic repulsions effecting hydrolysis by the catalytic glutamate
residue. The latter is consistent with the observed values of kcat, which is significantly
enhanced while the KM values are in a similar range. Additionally, WT MilB less
efficiently hydrolyzes dCMP possibly because the 2′ꢀgroup of dCMP cannot form any
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such hydrogen bonding interaction to orient it for catalysis. Thus, WT MilB catalytic
residues are best positioned for hydrolysis of CMP with the aid of the 2′ꢀhydroxyl group
orienting CMP in the binding pocket. These observations would also apply to WT and
F6Y BcmB, which show patterns similar to WT and F17Y MilB in efficiency for
hydrolysis of CMP and dCMP.
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Supplemental Information
Table S1 and Figures S1 and S2. This material is available free of charge at
http://pubs.acs.org.
The authors declare no competing financial interest.
Acknowledgements
We thank Dr. Cynthia Kinsland for cloning MilB and BcmB, Leslie Kinsland for
assistance in preparing the manuscript. We thank the staff of the NEꢀCAT at the APS for
assistance with the data collection.
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REFERENCES
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1.
Li, L., Xu, Z., Xu, X., Wu, J., Zhang, Y., He, X., Zabriskie, T. M., and Deng, Z.
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Table 1: Summary of data collection and refinement statistics.
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SeMet MilBꢀSO4
APS 24ꢀIDꢀC
1.95
MilBꢀCMP
APS 24ꢀIDꢀC
1.55
BcmBꢀPO4
APS 24ꢀIDꢀC
2.99
beamline
resolution (Å)
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
wavelength (Å)
space group
0.97918
0.97918
0.97918
P6222
C2
C2
a (Å)
b (Å)
97.8
97.8
45.2
100.4
177.9
40.2
c (Å)
β (°)
61.9
90.0
2.1
41
1
71.5
99.6
2.1
41
2
98
98.0
3.2
61
3
Matthews coefficient
% solvent
mol/asu
Measured reflections
Unique reflections
Average I/σ
Redundancy
Completeness (%)
Rsym (%)b
No. of protein atoms
No. of ligand atoms
No. of water atoms
137124
23697
37.1 (5.3)a
5.7 (5.4)
98 (97)
162974
44996
25.1(3.6)
3.6(3.6)
99.8 (100)
54150
14604
17.8 (4.7)
3.7(3.8)
99.9(100)
5.4 (32.1)
1164
10
5.7 (35.2)
2369
41
10.0(34.2)
2700
15
96
196
10
Reflections in working set
Reflections in test set
Rꢀfactor/Rfree (%)c
23686
1168
20.6/25.2
42663
2269
19.9/22.7
14582
737
20.7/23.9
rms deviation from ideals
bonds (Å)
0.007
1.072
0.006
1.061
0.015
1.975
angles (°)
average B factor for protein (Å2)
average B factor for water (Å2)
28.1
34.1
31.3
18.0
25.6
17.0
49.1
54.5
84.2
average B factor for ligand (Å2)
Ramachandram plot
most favored (%)
97.3
2.7
0.0
98.0
2.0
0.0
96.3
3.7
0.0
allowed (%)
disallowed (%)
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