Year: 2007
Type: article
Abstract: Bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood because of a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive, and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls. Bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood because of a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive, and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls. The characteristic shape of a bacterial cell is a function of the three-dimensional architecture of the cell wall (1Höltje J.V. Microbiol. Mol. Biol. Rev. 1998; 62: 181-203Crossref PubMed Google Scholar). The major rigid component of the cell wall is peptidoglycan, a mesh-like polymer comprising linear glycan chains held together by peptide cross-links (2van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar). The glycan chains are assembled from a diphospholipid-linked disaccharide-peptide precursor, Lipid II, by peptidoglycan glycosyltransferases (PGTs) 3The abbreviations used are: PGT, peptidoglycan glycosyltransferase; TP, transpeptidase; PBP, penicillin-binding protein; TEMED, N, N, N', N'-tetramethylethylenediamine; PG, peptidoglycan; NAG, N-acetylglucosaminyl; NAM, N-acetylmuramyl. (Fig. 1). These glycan chains are then cross-linked via the attached peptides by transpeptidases (TPs) (3Macheboeuf P. Kim C. Job V. Dideberg O. Dessen A. FEMS Microbiol. Rev. 2006; 30: 673-691Crossref PubMed Scopus (338) Google Scholar, 4van Heijenoort J. Nat. Prod. Rep. 2001; 18: 503-519Crossref PubMed Scopus (369) Google Scholar). The transpeptidases are targets for several families of clinically important antibiotics, including the penicillins, cephalosporins, and imipenems (5Fisher J.F. Kim S.O. Mobashery S. Chem. Rev. 2005; 105: 395-424Crossref PubMed Scopus (771) Google Scholar, 6Kahne D. Kim C. Lu W. Walsh C. Chem. Rev. 2005; 105: 425-448Crossref PubMed Scopus (536) Google Scholar), and the PGTs are thought to be promising antibacterial targets as well. There is a potent family of natural products, represented by the phosphoglycolipid moenomycin, which inhibit the PGTs with lethal cellular effects (7Ostash B. Kim A. Walker S. Chem. Biol. 2007; 14: 257-267Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 8Ostash B. Kim S. Curr. Opin. Chem. Biol. 2005; 9: 459-466Crossref PubMed Scopus (55) Google Scholar, 9Taylor J.G. Kim X. Oberthur M. Zhu W. Kahne D.E. J. Am. Chem. Soc. 2006; 128: 15084-15085Crossref PubMed Scopus (69) Google Scholar, 10Welzel P. Chem. Rev. 2005; 105: 4610-4660Crossref PubMed Scopus (68) Google Scholar). However, there are still no drugs in clinical use that operate by binding directly to the PGTs. A better under-standing of the PGTs and how they can be inhibited is required to exploit them as antibiotic targets and may also shed light on their cellular functions. PGTs are membrane-anchored polymerases that exist naturally in two forms, as N-terminal domains in bifunctional penicillin-binding proteins (PBPs), called class A high molecular weight PBPs, which contain C-terminal TP domains, and as monofunctional enzymes that do not contain transpeptidase domains (Fig. 1A) (2van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar, 11Lovering A.L. Kim L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (244) Google Scholar, 12Spratt B.G. Kim J. Taylor M. Merrick M.J. Mol. Microbiol. 1996; 19: 639-640Crossref PubMed Scopus (34) Google Scholar, 13Terrak M. Kim M. J. Bacteriol. 2006; 188: 2528-2532Crossref PubMed Scopus (45) Google Scholar). All PGTs contain five conserved motifs comprised of a number of invariant residues (14Goffin C. Kim J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar). Notable progress toward understanding PGTs has been made recently. For example, structures of two PGTs, one with moenomycin bound in the active site cleft, are now available and provide a starting point for inhibitor design (11Lovering A.L. Kim L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (244) Google Scholar, 15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar). However, we still lack answers to fundamental questions about the reactions catalyzed by PGTs, which limits our ability to think about their roles in bacterial cell growth and division. Questions that remain unanswered include the following. How long are the glycan chains made by particular PGTs? Do different PGTs within the same organism show significant differences in glycan product distributions? Are the glycan chain length distributions compatible with current models for the three-dimensional structure of the bacterial cell wall? Do other cellular factors affect chain length distributions? One of our goals is to develop facile in vitro methods to analyze PGT reactions so that these and other fundamental questions about their behavior can be addressed. PGTs catalyze the conversion of the peptidoglycan precursor, Lipid II, into glycan chains. The process involves the coupling of Lipid II units to form a tetrasaccharide, Lipid IV (Fig. 1, n = 1), followed by successive addition of Lipid II units to the reducing end of the growing polymer (16Perlstein D.L. Kim Y. Wang T.-S. Kahne D. Walker S. J. Am. Chem. Soc. 2007; (in press)PubMed Google Scholar). Understanding the PGT reaction requires having both Lipid II and Lipid IV substrates, combined with an assay that reports on glycan chain lengths. We and others have developed methods to obtain useful quantities of Lipid II and Lipid IV substrates (17Breukink E. Kim H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J. de Kruijff B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 18Schwartz B. Kim J.A. Wang Y. J. Am. Chem. Soc. 2001; 123: 11638-11643Crossref PubMed Scopus (118) Google Scholar, 19VanNieuwenhze M.S. Kim S.C. Zia-Ebrahimi M. Winger B.E. Hornback W.J. Saha S.L. Aikins J.A. Blaszczak L.C. J. Am. Chem. Soc. 2002; 124: 3656-3660Crossref PubMed Scopus (108) Google Scholar, 20Ye X.Y. Kim M.C. Brunner L. Walker D. Kahne D. Walker S. J. Am. Chem. Soc. 2001; 123: 3155-3156Crossref PubMed Scopus (154) Google Scholar, 21Zhang Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar); therefore, the necessary starting materials to study PGTs are now available (Fig. 2). In this paper, we describe a facile assay to analyze PGT reactions that provides information on reaction rates as well as glycan chain lengths. This assay has enabled us to compare two PGT domains from different organisms, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli, with respect to their ability to utilize disaccharide and tetrasaccharide substrates. For the A. aeolicus PGT domain, for which we recently reported the structure (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar), we have also carried out a comprehensive mutational analysis of the conserved residues in the five signature motifs that typify PGT domains (14Goffin C. Kim J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar, 22Terrak M. Kim T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (148) Google Scholar). We identify the most important residues for activity, which is pertinent to interpreting the crystal structures of PGTs, and we also show that the gel electrophoresis assay provides information about the products of mutant enzymes that cannot be obtained using other types of assays. The methods reported here provide new insights into the reactions catalyzed by PGTs and should make it possible to address many questions about how these enzymes behave. Information on the in vitro of PGTs, both and in with other of the peptidoglycan be useful for understanding the roles of PGTs in bacterial and division. and obtained from by the II as described by X.Y. Kim M.C. Brunner L. Walker D. Kahne D. Walker S. J. Am. Chem. Soc. 2001; 123: 3155-3156Crossref PubMed Scopus (154) Google and IV as described by Zhang Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar). A and from the and as described M. Kim Y. C. B. J.V. Kahne D.E. J. Am. Chem. Soc. 2006; 128: PubMed Scopus Google Scholar). used for reactions obtained from electrophoresis from from The obtained from All other and from and of PGT and of the PGT of A. aeolicus and E. have been described (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar, 21Zhang Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar). The PGT of E. PBP1A obtained as The the E. PBP1A from E. PBP1A using the are in The product then and into the and of to The by and followed in 21Zhang Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google with described in the of the PGTs for substrates and aeolicus in a new of used to make the from the using the in the and of carried out as reported in 15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google for the of as described by A.J. Kim J. PubMed Scopus Google and Kim PubMed Scopus Google with the to with and gel using the gel in a of in gel is the of both and and the of to and the addition of by the reaction to and them in of then the as with than on a II gel current with a of V. for the from the end of the and by using a and carried out with the using the gel analysis The in the of the of also by SDS-PAGE of a Lipid II and Lipid IV reaction in (Fig. with PGT reactions using enzymes from the A. aeolicus on and with a to in a Reactions with from E. All reactions with moenomycin A and on gel II used as a reactions carried out in A and Reactions and for the A. aeolicus PGT and and for the E. PGT IV used as a substrate with E. PGT domain, as reactions A with and PGT and and For A. PGT reactions A with and PGT and 1, and of with Lipid by E. PBP1A with in A with for for the reaction and by for The the into One of starting as a Reactions in of starting and of E. PBP1A A. aeolicus PGT in the of Lipid II for A by E. PBP1A and II in for in A using Lipid II and and analysis using a assay as described (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar, L. Kim D. B. Y. Walker S. Kahne D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). the reactions and analysis then using the SDS-PAGE assay described a for the of to monitor PGT on the of products B. Kim J.A. Wang Y. J. Am. Chem. Soc. 2001; 123: 11638-11643Crossref PubMed Scopus (118) Google Scholar, Kim J.V. PubMed Scopus Google Scholar, J. Kim A. J. Y. 2006; PubMed Scopus Google Scholar, Kim PubMed Scopus Google Scholar, B. Kim J.A. Wang Y. 2002; PubMed Scopus Google Scholar), but cannot different glycan chain which limits the information they provide about the the glycan products of a PGT reaction are of disaccharide units that have a we gel electrophoresis methods to analyze glycan chain length Lipid II (Fig. and and Lipid IV (Fig. substrates as described X.Y. Kim M.C. Brunner L. Walker D. Kahne D. Walker S. J. Am. Chem. Soc. 2001; 123: 3155-3156Crossref PubMed Scopus (154) Google Scholar, 21Zhang Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar), and via has that substrates with the of the as on the and have to substrates Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar, B. Kim J.A. Wang Y. 2002; PubMed Scopus Google Scholar). high the A. aeolicus PBP1A PGT the substrates into of products a of glycan chain lengths. gel for PGT products utilize a A.J. Kim J. PubMed Scopus Google Scholar, Kim PubMed Scopus Google Scholar). The of Lipid II the the progress of the to be A gel for glycan from one to units as from the using the substrates (Fig. also The of glycan chains is to the of the used in reaction and other because glycan chains are is The use of substrates in the reaction the for to it should be possible to this assay to products of reactions carried out with substrates in other with on the peptide chains. with Lipid II as the an assay in to glycan we the product distributions for the PGT domains of A. aeolicus PBP1A (Fig. and E. PBP1A (Fig. using the Lipid II substrate and an of There is a in the of products, which is for the PGT of E. This has also been for E. using other L. Kim D. B. Y. Walker S. Kahne D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, B. Kim J.A. Wang Y. 2002; PubMed Scopus Google and has been to a coupling to form a to a to the active the long glycan chains significant of products, with a processive mechanism in which coupling of Lipid II can of the The of glycan chains is for the E. PBP1A PGT than for the A. aeolicus PGT products are in the reaction for the E. than for the A. aeolicus PGT These results that the E. PGT is more processive than the A. aeolicus PGT The SDS-PAGE method described here should make it possible to differences in and glycan chain length exist different PGTs in the of the same Lipid recently reported that E. PBP1A can Lipid IV to products Y. Kim E.J. Wang T.S. Barrett D. Walker S. Kahne D.E. J. Am. Chem. Soc. 2007; 129: 3080-3081Crossref PubMed Scopus (66) Google Scholar). This because it thought that the disaccharide substrate (Lipid of with the growing glycan the assay used not provide information about glycan chain we have used the developed SDS-PAGE assay to the product for the A. aeolicus PBP1A (Fig. and E. PBP1A (Fig. PGT domains with the tetrasaccharide substrate For both this substrate more than Lipid II high In the of the tetrasaccharide products longer products a with a mechanism in which products are coupling In Lipid II to form long not with Lipid II and A. aeolicus PBP1A PGT E. PBP1A 1, starting from IV starting with starting with and Lipid II for are by reaction using II with E. PBP1A assay for of that PGT domains can Lipid IV as the substrate the that PGTs may longer than Lipid address this we a of from IV to use as starting materials (Fig. A and 1). The then with the PGT from A. aeolicus PBP1A (Fig. with E. PBP1A 2). in the of in the of PGT (Fig. A and compare and of glycan chains longer than Lipid However, the into longer products Lipid II to the reactions (Fig. A and as by the of new starting materials in A and and a of the of starting material. These results show that glycan units are of binding to PGTs and the are by a disaccharide units reaction in which Lipid II used as the substrate long glycan chains (Fig. we have that and of long glycan is than and processive of new chains. of show structural to (11Lovering A.L. Kim L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (244) Google Scholar, 15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar), an that the of The coupling reaction is to catalysis. There are two conserved glutamate residues in PGTs, one in in the A. aeolicus PGT and the other in in the A. aeolicus PGT have been to roles in (11Lovering A.L. Kim L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (244) Google Scholar, 22Terrak M. Kim T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (148) Google Scholar, J. Kim A. J. Y. 2006; PubMed Scopus Google Scholar, B. Kim J.A. Wang Y. 2002; PubMed Scopus Google Scholar, Kim J.V. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), for the of the glutamate is There is also a conserved and a conserved because most PGTs to be to a number of the conserved residues in the five invariant domains have been mutated to their in catalysis. the PGT of A. aeolicus PBP1A can be and in active form and for a of it is a for mutational analysis (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar). we of the conserved residues in the A. aeolicus PGT with with more All be in form and with the that of the with The of the mutant enzymes with that of the using a assay that on of products from Lipid II (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar, L. Kim D. B. Y. Walker S. Kahne D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). The substrate used in these which is the for the (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar). All with the (Fig. with the of the mutated including with chain and the reaction (Fig. that the of these is than the All other significant activity, that these other residues more roles in the We that the M. Kim T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (148) Google Scholar, J. Kim A. J. Y. 2006; PubMed Scopus Google on the of the conserved glutamate in the is a function of the different that is not this the mutant is active than the (Fig. These results out an for this glutamate in the mechanism (11Lovering A.L. Kim L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (244) Google and that it a in substrate that in the assay high and the product distributions using the SDS-PAGE assay (Fig. The and (Fig. and also turnover for and both and the more (Fig. and on the mutational we have that of the mutated residues and important roles in function because to to to assay by One of these is important because no turnover is for the highly (Fig. We from the that is the most important for and that the chain is The and of the gel electrophoresis assay it possible to of coupling products and provides more information than the this SDS-PAGE assay is of information about an is of in vitro is fundamental to about it may do in we have an SDS-PAGE method that us to monitor the of the glycan chain products of in vitro PGT reactions. of the glycan by gel electrophoresis for the analysis of the product distributions of PGTs. this SDS-PAGE we have the products by two different PGTs with two different substrates, Lipid II and Lipid Lipid II is the with a to than Lipid Lipid II also of the growing polymer to yield long glycan products with of In Lipid IV in a A crystal structure of the A. aeolicus PGT that a may of the active site the and we have that this to of the product from the active site (15Yuan Y. Kim D. Zhang Y. Kahne D. Sliz P. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5348-5353Crossref PubMed Scopus (124) Google Scholar). the coupling the product can in the active site and of coupling it can of the product of Lipid IV coupling to the new for of reaction may be than for Lipid in the of Lipid IV with the growing polymer may be than for Lipid A in the of coupling with the of make more to to the of products Lipid IV is the substrate. The that Lipid IV can function as a but longer that PGTs the of the as well as the of both the and the reducing end of the may the for Lipid II Lipid IV as a substrate. The results reported have for the of new disaccharide into the cell models for how this process may have been (2van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar). In one chains by PGTs are to transpeptidases for into the of the cell in the the disaccharide units are directly into the of the cell wall in vitro results show that PGTs Lipid II to long but are also of and long that both models are the in vitro reported here show that the processive of long glycan chains from Lipid II is than the addition of Lipid II to long that the of peptidoglycan involves the of chains into the of the cell In different PGTs to be required different in the cell it is not there are differences in their The results here show that there are differences in in PGTs from different is possible that there are also differences in in PGTs from the same there may be differences in the of product with more processive PGTs longer The assay described here make it possible to the different PGTs show significant differences in this in with information on the and of the their different cellular This assay also of the factors that affect product lengths. For example, it be possible to the effects of other proteins domains that are in peptidoglycan on the length of the glycan chains that are also be possible to how enzymes to substrate product lengths. These types of are important because glycan polymer length has for the structure of bacterial the two models are one in which the chains are to the cell and one in which the chains are to the cell W. Kim J.V. J. Bacteriol. PubMed Scopus Google Scholar). The product in this using Lipid II as a are long to be compatible with the the is to of the bacterial cell the there are other factors in cells that the of the glycan This assay provides a to how PGT product length distributions are to other including in substrate and the of other of the We and for and with
Source: Journal of Biological Chemistry
Institutions: Harvard University, Baim Institute for Clinical Research
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2086345344