Crystal Structure of Streptococcus pneumoniae N-Acetylglucosamine-1-phosphate Uridyltransferase Bound to Acetyl-coenzyme A Reveals a Novel Active Site Architecture

The bifunctional bacterial enzymeN-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against β-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. TheS. pneumoniae GlmU molecule is organized in two separate domains connected via a long α-helical linker and associates as a trimer, with the 50-Å-long left-handed β-helix (LβH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LβH core and exchanged with the β-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LβH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits. The bifunctional bacterial enzymeN-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against β-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. TheS. pneumoniae GlmU molecule is organized in two separate domains connected via a long α-helical linker and associates as a trimer, with the 50-Å-long left-handed β-helix (LβH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LβH core and exchanged with the β-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LβH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits. GlmU catalyzes acetyltransfer from acetyl-coenzyme A (AcCoA)1 to glucosamine-1-P with release of GlcNAc-1-P, and subsequently uridyltransfer from UTP to GlcNAc-1-P in the presence of Mg2+, yielding PPi and the nucleotide-activated precursor sugar UDP-GlcNAc (1Mengin-Lecreulx D. van Heijenoort J.J. J. Bacteriol. 1993; 175: 6150-6157Crossref PubMed Google Scholar) (see Fig.1 A). UDP-GlcNAc is one of the main cytoplasmic precursors of the bacterial cell wall, being situated at the branch point of two important biosynthetic pathways, namely peptidoglycan and lipid A biosynthesis (2Raetz C.R.H. Neidhardt F.C. Lin E. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1035-1063Google Scholar). In eukaryotes, a bifunctional enzyme equivalent to GlmU is missing, and acetyltransfer and uridyltransfer are accomplished by two distinct enzymes, both very distantly related in sequence to GlmU, the latter thus advancing to an attractive target for the development of new antibiotics. The crystal structures of a truncated form of Escherichia coli GlmU (GlmU-Tr) and of a GlmU-Tr·UDP-GlcNAc complex have been recently reported (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). These structures confirmed that the enzyme is organized in the following two separate domains as proposed previously (4Gehring A.M. Lees W.J. Mindiola D.J. Walsh C.T. Brown E.D. Biochemistry. 1996; 35: 579-585Crossref PubMed Scopus (87) Google Scholar, 5Mengin-Lecreulx D. van Heijenoort J. J. Bacteriol. 1994; 176: 5788-5795Crossref PubMed Scopus (167) Google Scholar): (i) an N-terminal uridyltransferase (PPase) domain, comprising Asn-3 to Arg-227, resembling the dinucleotide binding Rossmann fold, first reported in the lactate dehydrogenase family (6Rossmann M.G. Liljas A. Branden C.-I. Bansazak L.J. Boyer P.D. The Enzymes. Academic Press, New York1975: 61-102Google Scholar), and containing the signature motif G-X-G-T-(R/S)-(X)4-P-K, found in the majority of pyrophosphorylases, and (ii) a C-terminal acetyltransferase domain, containing the hexapeptide repeat (L/I/V)-(G/A/E/D)-X 2-(S/T/A/V)-X, a signature of the unusual left-handed β-helix (LβH) structural motif, typically found in other bacterial acetyl- and acyltransferases (7Vuorio R. Hirvas L. Vaara M. FEBS Lett. 1991; 292: 90-94Crossref PubMed Scopus (24) Google Scholar) (Fig. 1 B). Furthermore, the GlmU-Tr·UDP-GlcNAc complex structure identified the precise location of the uridyltransfer reaction, the pyrophosphorylase activity of GlmU-Tr being retained. However, acetyltransferase activity was lost because of spontaneous truncation during purification, confirming that the bifunctional enzyme possesses indeed two distinct active sites located in separate domains, with the acetyltransferase activity residing in the C-terminal portion of the enzyme (4Gehring A.M. Lees W.J. Mindiola D.J. Walsh C.T. Brown E.D. Biochemistry. 1996; 35: 579-585Crossref PubMed Scopus (87) Google Scholar). Although the crystal structure of the E. coli enzyme, coupled to mutagenesis studies, has revealed some residues crucial for pyrophosphorylase activity (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar), the catalytic machineries responsible for both pyrophosphorylase and acetyltransferase activity remain to be elucidated. Here we present the crystal structures of full-length GlmU from the pathogenic organism Streptococcus pneumoniae in its unbound form and in complex with AcCoA and both AcCoA and the product UDP-GlcNAc. These structures define the precise location of the acetyltransferase active site, reveal substantial conformational changes occurring both upon AcCoA and UPD-GlcNAc binding, and highlight the structural elements responsible for substrate recognition and catalysis in the two distinct active sites of this bifunctional enzyme. The coding region of SpGlmU was amplified from S. pneumoniae strain R 800 DNA by polymerase chain reaction and inserted into the bacterial expression plasmid PQE30 (Qiagen). Recombinant SpGlmU was overexpressed in M15 cells and purified to homogeneity by nickel-nitrilotriacetic acid-agarose and gel filtration chromatography. Enzyme activity has been tested and found similar to that of full-length E. coli GlmU (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). Crystals were grown at 20 °C by the hanging-drop vapor diffusion method by mixing equal volumes of protein solution (13 mg/ml) with reservoir solution composed of 26% (v/v) PEG 400, 50 mm NaCl, and 300 mmCaCl2 at pH 8.0 by TRIS-HCl. Small rhombohedral crystals with a typical size of 0.1 × 0.1 × 0.1 mm appeared within 1 week. Crystals belong to space group R3 and contain two molecules per asymmetric unit. As molecular replacement with GlmU-Tr (Protein Data Bank entry 1FXJ) failed, selenomethionine-substituted enzyme was produced using the same bacterial strain grown in minimum medium and supplemented, before induction, with selenomethionine and amino acids known to inhibit methionine biosynthesis (8Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). The yield of selenomethionine substitution was about 50% as judged by matrix-assisted laser desorption ionization/time of flight mass spectroscopy analysis. Crystals of bigger dimensions and higher diffraction quality were obtained for the selenomethionine-substituted enzyme under the same crystallization conditions as adopted for the native protein. Crystals for the AcCoA complex were obtained by incubating the enzyme with 20 mm AcCoA prior to crystallization and lowering the PEG 400 concentration to 18% (v/v). AcCoA·UDP-GlcNAc complex crystals were obtained by cocrystallization with 20 mm AcCoA followed by harvesting into a stabilizing solution made of 30% (v/v) PEG 400, 50 mm NaCl, 300 mm CaCl2 at pH 8.0 by TRIS-HCl and supplemented with 10 mm UDP-GlcNAc. All data sets were collected at 100 K on flash-frozen crystals. Cryosolutions were of the same composition as the crystallization/harvesting solutions with the addition of an increasing amount of PEG 400 and supplemented with 5% (v/v) glycerol. A 3-wavelength multiple anomalous dispersion data set for selenomethionine-substituted SpGlmU was collected on beamline BM14 (European Synchrotron Radiation Facility, Grenoble, France), a data set for native SpGlmU and data for the AcCoA complex were collected on beamlines ID14-EH2, and data for the AcCoA·UDP-GlcNAc complex were collected on beamline ID14-EH3 (European Synchrotron Radiation Facility, Grenoble, France). Data were indexed and integrated with DENZO (9Otwinowski Z. Minor W. Carter J.C.W. Sweet R.M. Methods in Enzymology. Academic Press, New York1997: 307-326Google Scholar), and all further computing was carried out with the CCP4 program suite (10CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760Crossref PubMed Scopus (19797) Google Scholar) unless otherwise stated. Data collection statistics are summarized in Table I and Table II.Table IMAD data collection and statisticsf ′′maxf ′minRemoteWavelength (Å)0.97870.97890.8856Resolution1-aValues in parentheses are for the highest resolution shell.(Å)20 - 2.8 (2.95 - 2.80)20 - 2.8 (2.95 - 2.80)20 - 2.8 (2.95 - 2.80)Rmerge1-aValues in parentheses are for the highest resolution shell.1-bRmerge = ΣhklΣi‖Ihkli − 〈Ihkli〉‖/ΣhklΣi〈Ihkli〉.8.2 (32.8)7.5 (30.6)7.7 (27.6)Ranom1-aValues in parentheses are for the highest resolution shell.1-cRanom = Σ‖〈I +〉 − 〈I −〉‖/Σ(〈I +〉 + 〈I −〉).6.8 (24.0)6.1 (22.2)4.9 (16.1)〈I/ςI〉1-aValues in parentheses are for the highest resolution shell.6.0 (1.4)6.5 (1.5)5.1 (1.9)Redundancy1-aValues in parentheses are for the highest resolution shell.3.7 (3.7)3.7 (3.7)4.7 (4.7)Completeness1-aValues in parentheses are for the highest resolution shell.(%)99.8 (99.8)99.8 (99.8)99.9 (99.8)Anomalous completeness1-aValues in parentheses are for the highest resolution shell.(%)99.2 (98.1)98.9 (97.5)99.7 (98.7)1-a Values in parentheses are for the highest resolution shell.1-b Rmerge = ΣhklΣi‖Ihkli − 〈Ihkli〉‖/ΣhklΣi〈Ihkli〉.1-c Ranom = Σ‖〈I +〉 − 〈I −〉‖/Σ(〈I +〉 + 〈I −〉). Open table in a new tab Table IIData collection and refinement statisticsApo-formAcCoA complexAcCoA·UDP-GlcNAc complexResolution2-aValues in parentheses are for the highest resolution shell.(Å)40.0–2.3 (2.37–2.30)40.0–2.5 (2.56–2.50)50.0–1.75 (1.80–1.75)Space group/cell dimensions (Å)R3/a = b = 92.71, c = 280.39R3/a = b = 92.46, c = 279.56R3/a = b = 89.51, c = 278.75No. of observations7996873418187478No. of unique reflections377582921882248Completeness2-aValues in parentheses are for the highest resolution shell.(%)95.1 (97.6)94.7 (96.6)97.9 (96.4)Redundancy2-aValues in parentheses are for the highest resolution shell.2.1 (1.5)2.5 (1.8)2.3 (2.0)〈I/ςI〉2-aValues in parentheses are for the highest resolution shell.5.2 (2.8)23.1 (10.8)12.3 (1.9)Rmerge2-aValues in parentheses are for the highest resolution shell.2-bRmerge = ΣhklΣi‖Ihkli − 〈Ihkli〉‖/ΣhklΣi〈Ihkli〉.8.0 (27.4)2.4 (6.9)4.6 (35.4)B from Wilson plot (Å2)48.6642.6218.37Protein atoms2-cPer asymmetric unit, corresponding to two molecules of SpGlmU.662669286928Water molecules/ligand atoms/Ca+22-cPer asymmetric unit, corresponding to two molecules of SpGlmU.185/−/6270/102/4694/180/8R cryst2-dRcryst = Σ∥F o‖ − ‖F c∥/Σ‖F o‖./R free(%)20.7/24.518.4/24.318.3/21.9r.m.s. 1–2 bond distances (Å)0.0120.0110.011r.m.s. 1–3 bond angles (°)1.651.561.53Average main chain/side chain B (Å2)48.1/53.035.7/38.020.7/24.6Average B AcCoA·UDP-GlcNAc (Å2)−/−44.5/−20.4/30.6r.m.s. B, main chain bonded atoms (Å2)1.20.961.1Ramachandran plot outliersnonenonenone2-a Values in parentheses are for the highest resolution shell.2-b Rmerge = ΣhklΣi‖Ihkli − 〈Ihkli〉‖/ΣhklΣi〈Ihkli〉.2-c Per asymmetric unit, corresponding to two molecules of SpGlmU.2-d Rcryst = Σ∥F o‖ − ‖F c∥/Σ‖F o‖. Open table in a new tab The SpGlmU structure was solved using the program SOLVE (11Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The initial multiple anomalous dispersion phases had a mean figure of merit of 0.340- to 2.8-Å resolution and were improved by density modification with the program DM (12Cowtan K. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar) and extended to the resolution of the native data set (2.3 Å). Because of the low yield of selenomethionine incorporation only a few of these residues could be located in the experimental electron density maps, which were of mediocre quality. Non-crystallography symmetry averaging and phase combination techniques were of great help in overcoming these problems, and a preliminary model could be constructed for most of the LβH and the core of the N-terminal using the program A. Cambillau C. Scholar). However, most of the some of the in the N-terminal domain, and the C-terminal residues out to be at A of the quality was for the AcCoA A model could be comprising residues A crystal upon of AcCoA complex crystals in the solution containing UDP-GlcNAc, and the structure was solved by molecular replacement with the program J. Acta Crystallogr. Sect. A. 1994; 50: Scopus Google Scholar). was carried out with the Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar) and P.D. J. M. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar), using the method and and symmetry of the were set during refinement for of the model and were with the program A. R. Biol. 1999; PubMed Scopus Google Scholar). The of the was with the program R. M. D. J. J. Crystallogr. 1993; Google Scholar). statistics are summarized in have been in the Protein Data Bank under for and and for the AcCoA and the AcCoA·UDP-GlcNAc respectively. Fig.1 B was with Protein 1993; PubMed Scopus Google Scholar), and were with The for Scholar) and Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: PubMed Scopus Google pyrophosphorylase of the of E. coli GlmU-Tr and with the pyrophosphorylase signature motif in The is on a of of the of the pyrophosphorylase B, of the of SpGlmU to AcCoA and AcCoA·UDP-GlcNAc with that between the two complex structures are for the form, and for the structure elements are of the binding site in the the molecule is as in A with the signature motif molecules are and the is are acetyltransferase of the in the LβH with the three in the in respectively. AcCoA and are The chain of the proposed catalytic and the of the and the C-terminal that form the are important residues are B, of the SpGlmU acetyltransferase active site with AcCoA and The of each subunit is as in for only the C-terminal of the third subunit is The of the catalytic and other important residues are The crystal structure of full-length SpGlmU was determined by multiple anomalous dispersion The and structures were refined to 2.3, 2.5, and 1.75 Å, and have The structure of residues to and to The located in the pyrophosphorylase domain, and the residues of the acetyltransferase domain, could be because of of electron The two complex and of to and electron density could be for both AcCoA and UDP-GlcNAc prior to the incorporation in the refinement (Fig. The SpGlmU molecule into a with dimensions of × × (Fig. The LβH domains are packed against each other in a parallel an α-helical linker on of each β-helix and the pyrophosphorylase far away from the The SpGlmU for the two and is similar to the complex with a mean of for (Fig. The complex in is to the complex structure in the acetyltransferase mean of for However, the two complex structures in the pyrophosphorylase domain, as further The SpGlmU for residues to is similar to enzyme (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). However, the of the pyrophosphorylase and the acetyltransferase between the crystal structures of SpGlmU and E. (Fig. the two GlmU structures present a in the of the α-helical that this in a A of this are between GlmU-Tr and SpGlmU occurring in the of the pyrophosphorylase neighboring the of the α-helical These conformational with a of the pyrophosphorylase domain, as to the acetyltransferase domain, that the structures only of a The SpGlmU be into two by the active site The first containing the sequence motif form the binding the responsible for recognition of the sugar the residues of the N-terminal (Fig. B). between the domains of and the complex mean of for that the enzyme a substantial conformational upon In the of UDP-GlcNAc and SpGlmU an in the UDP-GlcNAc complex two within the sugar binding each other giving rise to a (Fig. B). product binding the region residues as a a in a of the The of the of the the the following into an extended These the two to each that in the UDP-GlcNAc complex the (Fig. in the unbound form these two residues are that the two a of upon substrate binding and the sugar into the active site from The of the of SpGlmU could be for the E. coli GlmU-Tr enzyme, the crystal structures reveal a for both the and UDP-GlcNAc (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). However, of the crystal in the E. coli GlmU-Tr structures that the pyrophosphorylase is into its in both the and the GlmU-Tr·UDP-GlcNAc complex by the in SpGlmU crystals. The of the enzyme with the and the sugar are within the complex crystal structures from S. pneumoniae and E. coli GlmU, in the of the in the GlmU-Tr·UDP-GlcNAc complex both are in the complex the is to the of and located within the signature both a with and situated in the and in the of the long α-helical the of and thus the important with the of the (Fig. B). with the bond to that of stabilizing the product and have a in substrate recognition during the reaction P.A. J. Biol. PubMed Google Scholar), with mutagenesis data of coli GlmU enzyme (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). in the crystal structure of the E. complex the residues and are located far away from the These residues are carried by three structural elements in with each with the α-helical and with the acetyltransferase of a neighboring As the E. coli GlmU-Tr structures from the SpGlmU structures in the of the acetyltransferase and pyrophosphorylase domains, because of enzyme in E. coli the α-helical the signature motif away from the substrate binding of the pyrophosphorylase domain, which the in the of E. coli as with the enzyme (3Brown K. Pompeo F. Dixon S. Mengin-Lecreulx D. Cambillau C. Bourne Y. EMBO J. 1999; 18: 4096-4107Crossref PubMed Scopus (168) Google Scholar). The C-terminal acetyltransferase LβH an with the three by three parallel composed of B). The long β-helix of full-length SpGlmU of 10 the E. coli GlmU-Tr structure is truncated the The to a of a SpGlmU subunit upon formation is a in the highest with other LβH The of the is only at the by a the region to which from one of the of the and an adjacent subunit (Fig. B). The and of the SpGlmU is the of the C-terminal Although this is a within the family of bacterial a has been reported for a of and is to as D. Protein 50: PubMed Google Scholar). 10 the chain is exchanged with an adjacent subunit, thus the unique of an within the LβH with the α-helical linker on of the LβH domain, the C-terminal to the of the SpGlmU the the of the of a neighboring this point the chain between two neighboring and in the of the two and in with the of an adjacent long and very narrow is in this AcCoA located at the of two and from the by the exchanged C-terminal of the third subunit and the that the is for the acetyltransferase an active site architecture located at the junction of three is and an coupled to a a binding The C-terminal region could be in the that this region is and only upon AcCoA other structural upon AcCoA binding in the acetyltransferase domain, for the which is in the as by a main chain of as with an main chain of for the of the acetyltransferase of the AcCoA is by of the group between the of and from the group to and and with from and from the to atoms of and The is to the and with the protein. of the complex with other related bacterial a location of the AcCoA binding AcCoA a very similar to the for in the S.L. Biochemistry. PubMed Scopus Google Scholar), at the group and with an extended parallel to the LβH (Fig. A). Although the C-terminal from which the AcCoA binding site is and to other related bacterial of the C-terminal portion upon binding has been previously reported for S.L. Biochemistry. PubMed Scopus Google Scholar). AcCoA as substrate the the to a a to the substrate PubMed Scopus Google J. J. P.A. J. Biol. PubMed Scopus Google Scholar). In the of the first of these two the of the residues in the E. coli enzyme was by mutagenesis F. van Heijenoort J. Mengin-Lecreulx D. J. Bacteriol. PubMed Google Scholar). However, of the residues are between known GlmU and acetyltransferase activity was only by the of which is and the of the LβH in the E. coli GlmU-Tr SpGlmU only one located 10 from the active site, thus the of a enzyme of the SpGlmU active site a group on a catalytic by the residues and (Fig. B). is the only located in of the which as a the of glucosamine-1-P for of to the form of the one on located the is to with the of the on the at the The within bond to the could have a in at the end of the catalytic The of is by a of SpGlmU with the crystal structure of which SpGlmU to a proposed to as the S.L. Biochemistry. PubMed Scopus Google Scholar). A has been to as the as in the related hexapeptide acetyltransferase from M. S.L. Biochemistry. PubMed Scopus Google Scholar). In of a complex with we have into the containing the catalytic and by from two neighboring and the The of is by a of residues and for binding the a by that is a very substrate with for the acetyltransfer reaction (4Gehring A.M. Lees W.J. Mindiola D.J. Walsh C.T. Brown E.D. Biochemistry. 1996; 35: 579-585Crossref PubMed Scopus (87) Google Scholar). In model the amino group on is within binding from the proposed catalytic of and to a on the (Fig. B). The three crystal structures of SpGlmU in unbound and form in this highlight structural to the acetyltransferase reaction and define a structural to design new antibiotics. A of the two distinct GlmU catalytic mechanisms further of substrate and for the S. pneumoniae the of the Synchrotron Radiation for in data and and for acetyl-coenzyme A uridyltransferase truncated form of Escherichia coli GlmU pyrophosphorylase left-handed β-helix Streptococcus pneumoniae GlmU