Structure and Function of Sedoheptulose-7-phosphate Isomerase, a Critical Enzyme for Lipopolysaccharide Biosynthesis and a Target for Antibiotic Adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of l-glycero-d-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of d-sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism. The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of l-glycero-d-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of d-sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism. Lipopolysaccharide (LPS) 4The abbreviations used are: LPSlipopolysaccharideDTTdithiothreitolGln6Pglucosamine 6-phosphateGmhAsedoheptulose-7-phosphate isomeraseGmhBd-heptose-1,7-bisphosphate phosphataseHldEbifunctional d-β-d-heptose phosphate kinase/d-β-d-heptose 1-phosphate adenyltransferase; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acidMICminimal inhibitory concentrationOMouter membranePEGpolyethyleneS7Pd-sedoheptulose 7-phosphateSeMetselenomethionineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glyciner.m.s.d.root mean square deviationBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.4The abbreviations used are: LPSlipopolysaccharideDTTdithiothreitolGln6Pglucosamine 6-phosphateGmhAsedoheptulose-7-phosphate isomeraseGmhBd-heptose-1,7-bisphosphate phosphataseHldEbifunctional d-β-d-heptose phosphate kinase/d-β-d-heptose 1-phosphate adenyltransferase; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acidMICminimal inhibitory concentrationOMouter membranePEGpolyethyleneS7Pd-sedoheptulose 7-phosphateSeMetselenomethionineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glyciner.m.s.d.root mean square deviationBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. is an essential component of the outer membrane in Gram-negative bacteria (1Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar). LPS not only functions as a protective barrier preventing cell entry of hydrophobic molecules, including bile salts, detergents, and lipophilic antibiotics, but also helps maintain the structural integrity of the outer membrane. Thus, LPS is vital for bacterial virulence and antibiotic sensitivity in pathogenic Gram-negative bacteria. lipopolysaccharide dithiothreitol glucosamine 6-phosphate sedoheptulose-7-phosphate isomerase d-heptose-1,7-bisphosphate phosphatase bifunctional d-β-d-heptose phosphate kinase/d-β-d-heptose 1-phosphate adenyltransferase; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid minimal inhibitory concentration outer membrane polyethylene d-sedoheptulose 7-phosphate selenomethionine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine root mean square deviation 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. lipopolysaccharide dithiothreitol glucosamine 6-phosphate sedoheptulose-7-phosphate isomerase d-heptose-1,7-bisphosphate phosphatase bifunctional d-β-d-heptose phosphate kinase/d-β-d-heptose 1-phosphate adenyltransferase; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid minimal inhibitory concentration outer membrane polyethylene d-sedoheptulose 7-phosphate selenomethionine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine root mean square deviation 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Gram-negative pathogens are increasingly becoming a serious clinical threat. Multidrug-resistant hospital-acquired infections caused by enteric bacteria such as Escherichia coli and Klebsiella pneumoniae, and by emerging pathogens of environmental origin such as Acinetobacter baumannii and Pseudomonas aeruginosa, are the next big problem facing the infectious disease community. Furthermore, Gram-negative pathogens of animal origin such as E. coli O157-H7 are ongoing threats to agriculture and water quality. New chemotherapeutic strategies against Gram-negative bacteria are therefore required. LPS biosynthesis represents a unique Gram-negative target for new antimicrobial intervention. LPS comprises lipid A, a core oligosaccharide, and in some bacteria, an O-specific polysaccharide chain. The core oligosaccharide has an inner core region consisting of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and one or more heptose units, and an outer core, consisting of additional sugar residues (Fig. 1A) (reviewed in Refs. 1Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar, 2Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2838) Google Scholar, 3Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 4Yethon J.A. Whitfield C. Curr. Drug Targets Infect. Disord. 2001; 1: 91-106Crossref PubMed Scopus (71) Google Scholar). Lipid A and Kdo are highly conserved in Gram-negative bacteria and essential for cell viability. The biosynthesis of these molecules is therefore a target for traditional antibiotic discovery efforts. Indeed, small molecule inhibitors of lipid A biosynthesis have been reported to have anti-Gram-negative activity (5Onishi H.R. Pelak B.A. Gerckens L.S. Silver L.L. Kahan F.M. Chen M.H. Patchett A.A. Galloway S.M. Hyland S.A. Anderson M.S. Raetz C.R. Science. 1996; 274: 980-982Crossref PubMed Scopus (355) Google Scholar). Most Gram-negatives also contain one or more l-glycero-d-manno-heptose molecules attached to the Kdo. Mutants in heptose metabolism, which are viable in laboratory conditions, are avirulent and highly susceptible to antibiotics (reviewed in Ref. 6Valvano M.A. Messner P. Kosma P. Microbiology. 2002; 148: 1979-1989Crossref PubMed Scopus (112) Google Scholar). Heptose biosynthesis is thus a non-traditional target for Gram-negative selective antimicrobial agents. Inhibitors of heptose biosynthesis could be used as anti-virulence drugs or could be co-administered with antibiotics that do not normally cross the outer membrane barrier (e.g. novobiocin and erythromycin) to sensitize bacteria to these agents. We have termed such molecules antibiotic adjuvants (7Wright G.D. Sutherland A.D. Trends Mol. Med. 2007; 13: 260-267Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The outer core carbohydrates and the O-specific polysaccharide side chains, also known as O-antigens, comprise the remainder of the LPS polymer. These components vary significantly by organism (1Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar). They are not essential for cell growth but do mediate host-microbe interactions and play a significant role in virulence. Inhibitors of outer core and O-antigen biosynthesis could, therefore, be strategically deployed as organismspecific anti-virulence compounds. All levels of LPS biosynthesis represent underexploited targets for new anti-microbial agents. The heptose biosynthetic pathway in Gram-negative bacteria, in particular, is highly attractive being essential for virulence and antibiotic sensitivity. Heptoses targeted to the inner core LPS are synthesized within the cytosol as ADP-activated l-glycero-β-d-manno-heptose molecules (8Kneidinger B. Marolda C. Graninger M. Zamyatina A. McArthur F. Kosma P. Valvano M.A. Messner P. J. Bacteriol. 2002; 184: 363-369Crossref PubMed Scopus (147) Google Scholar, 9Eidels L. Osborn M.J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1673-1677Crossref PubMed Scopus (66) Google Scholar, 10Kneidinger B. Graninger M. Puchberger M. Kosma P. Messner P. J. Biol. Chem. 2001; 276: 20935-20944Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Biosynthesis is initiated from d-sedoheptulose 7-phosphate (S7P). Sedoheptulose-7-phosphate isomerase (GmhA) catalyzes the first committed step in the pathway (Fig. 1B) (11Brooke J.S. Valvano M.A. J. Bacteriol. 1996; 178: 3339-3341Crossref PubMed Google Scholar, 12Brooke J.S. Valvano M.A. J. Biol. Chem. 1996; 271: 3608-3614Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Eidels L. Osborn M.J. J. Biol. Chem. 1974; 249: 5642-5648Abstract Full Text PDF PubMed Google Scholar). In E. coli, phosphorylation at the 1 position of the resulting in d-glycero-α,β-d-manno-heptose 7-phosphate is then catalyzed by the kinase moiety of the bifunctional d-β-d-heptosephosphate kinase/d-β-d-heptose-1-phosphate adenyltransferase (HldE) (14McArthur F. Andersson C.E. Loutet S. Mowbray S.L. Valvano M.A. J. Bacteriol. 2005; 187: 5292-5300Crossref PubMed Scopus (31) Google Scholar). A bifunctional HldE is also predicted in the opportunistic pathogen P. aeruginosa based on genomic sequence comparisons. However, in other pathogenic organisms, such as Burkholderia cenocepacia, this bifunctional enzyme is replaced by two distinct enzymes, HldA and HldC, which accomplish the respective functions (6Valvano M.A. Messner P. Kosma P. Microbiology. 2002; 148: 1979-1989Crossref PubMed Scopus (112) Google Scholar, 15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (109) Google Scholar). d-α,β-d-Heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the removal of the phosphate at the 7 position, whereas the adenyltransferase action of HldE (or mono-functional HldC) transfers the AMP moiety from ATP to give ADP-d-glycero-β-d-manno-heptose (6Valvano M.A. Messner P. Kosma P. Microbiology. 2002; 148: 1979-1989Crossref PubMed Scopus (112) Google Scholar, 16Valvano M.A. Marolda C.L. Bittner M. Glaskin-Clay M. Simon T.L. Klena J.D. J. Bacteriol. 2000; 182: 488-497Crossref PubMed Scopus (50) Google Scholar). Finally, ADP-d-β-D heptose epimerase (HldD) catalyzes the formation of ADP-l-glycero-β-d-manno-heptose, the precursor for the incorporation of heptose into the inner core, which is mediated by specific heptosyltransferases (17Sirisena D.M. MacLachlan P.R. Liu S.L. Hessel A. Sanderson K.E. J. Bacteriol. 1994; 176: 2379-2385Crossref PubMed Google Scholar, 18Morrison J.P. Tanner M.E. Biochemistry. 2007; 46: 3916-3924Crossref PubMed Scopus (17) Google Scholar). A key step in ADP-heptose biosynthesis is S7P isomerization catalyzed by GmhA. Previous studies of GmhA predicted its function using gene deletion and product analysis (12Brooke J.S. Valvano M.A. J. Biol. Chem. 1996; 271: 3608-3614Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Eidels L. Osborn M.J. J. Biol. Chem. 1974; 249: 5642-5648Abstract Full Text PDF PubMed Google Scholar). Mutation of gmhA also results in a compromised OM, effectively removing the protective barrier normally afforded by LPS, therefore greatly increasing susceptibility to antibiotics (11Brooke J.S. Valvano M.A. J. Bacteriol. 1996; 178: 3339-3341Crossref PubMed Google Scholar). Understanding the structure and function of GmhA could aid in the future development of inhibitors that would increase the permeability of Gram-negative pathogens and act synergistically with known antibiotics as a novel treatment for Gramnegative infections. We report crystal structures of E. coli and P. aeruginosa GmhA in apo, substrate, and product-bound forms and the use of this structural data to guide site-directed mutagenesis studies that enable prediction of the molecular mechanism of S7P isomerization, a potential target for new antimicrobial agents. Purification of GmhA—Purification of E. coli GmhA a Valvano M.A. G.D. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). additional step for GmhA used in and these GmhA to a and using a 1 dithiothreitol GmhA 1 only GmhA and against and P. aeruginosa GmhA in E. coli an and for and for in M.S. PubMed Scopus Google for at and at in with 1 and and in through and acid GmhA in and using a to a J. J. PubMed Scopus Google Scholar, M. J. Biochem. PubMed Scopus Google Scholar). to of GmhA at using the E. coli GmhA with an of polyethylene and and against of S7P at a concentration of 1 and replaced with to in and GmhA in a or P. aeruginosa in a of and and with a of and GmhA in a of S7P and and with All data at E. coli and GmhA data with an on an and using Biol. PubMed Scopus Google Scholar). for molecular using A. A. J. Scopus Google from GmhA J. S. 2006; PubMed Scopus Google GmhA by molecular using the E. coli structure as a P. aeruginosa GmhA data at the of the whereas product-bound GmhA data at the of the These data with 276: Scopus Google Scholar). J. Biol. PubMed Scopus Google in the Biol. 2000; PubMed Scopus Google then used to an the structure of product-bound P. aeruginosa the structure of the P. aeruginosa GmhA used as a and for GmhA structures using M. A. PubMed Scopus Google P. Biol. PubMed Scopus Google A. E. Proc. or P. J.S. J. M. Biol. PubMed Scopus Google and within using F. L. 2002; PubMed Scopus Google Scholar). using data and in for the are in for the are in for the are in for the are in for the are in and for the are in of for the are in in a new GmhA in E. coli gmhA in and using the site-directed mutagenesis of are in by sequence analysis using coli GmhA and molecular in by analysis using a to and into a cell with a to for at of and The data at and using the and GmhA specific and using a by and then to a using the to the of for synthesized from and based on the by and with S. A. M. C. J. Mol. Scopus Google Scholar). E. coli as Valvano M.A. G.D. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). acid of from E. coli by using a using and activity using a enzyme F. Biochemistry. PubMed Scopus Google Scholar). acid in the of S7P and using and and E. coli GmhA activity by product formation to HldE and and as Valvano M.A. G.D. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google with the The of of of of and of in a of initiated with of S7P for from to to 1 using GmhA in and used to E. coli M. M. M. H. Mol. Biol. 2006; Scopus Google to and by the into E. coli and E. coli at in minimal GmhA 1 of in of 1 of by and by to a membrane. GmhA using and used in inhibitory of novobiocin as as to and 1 in at in in the of of novobiocin to as the concentration of novobiocin to the of to of the in the absence of LPS coli and at for on minimal LPS from these as C.L. P. E. S. Valvano M.A. Mol. Biol. 2006; Google Scholar). LPS by in the and by C.L. P. E. S. Valvano M.A. Mol. Biol. 2006; Google Scholar, C.E. Biochem. PubMed Scopus Google Scholar). in of in using of acid for of acid for for of of of of for for and of of of in development by in of crystal structures of from E. coli and P. aeruginosa to and The structure from E. coli molecular using an based on the GmhA structure from E. coli in the with molecules of GmhA in as in A, and of using the E. J. Mol. Biol. 2007; PubMed Scopus Google that GmhA would in as a by studies for residues in or and therefore these are as in in the one at and two and at These are of a or of to in increased GmhA activity The and are resulting from interactions and and are and are through and interactions with The to and of and from P. aeruginosa in and the structure to using and The to and of and with the structure from E. coli, a of GmhA in the and residues in The of these could be with an of GmhA of a by (Fig. a are of and on one side and and on the side of the with and The is to the and is to GmhA structures from and J. S. 2006; PubMed Scopus Google Scholar). In to of we also structures of GmhA in the of and The structure of E. coli GmhA in with S7P to in a with The to and of and The the and from the of on the and which in the of (Fig. GmhA isomerase used to these and to a of product and is to have been during crystal additional at only one of the potential within the GmhA in this is with the of the to which this structure structural and analysis to the of of structures of and product-bound GmhA. A, E. coli GmhA in with P. aeruginosa GmhA in with d-glycero-d-manno-heptose All acid side with or product are and are The product-bound structure of GmhA from P. aeruginosa in to These of GmhA with In this to product in region (Fig. In to the and structures of E. coli the product-bound as a in the (Fig. However, by two from a could be (Fig. GmhA used in of these would that to an for distinct conformations of GmhA of or structures of GmhA have been and E. coli and product-bound P. aeruginosa and C. GmhA. in these structures be into two distinct and The E. coli structures as as the P. aeruginosa and the structures an whereas the P. aeruginosa and C. in the the and conformations are a new in the product-bound structure is in of the in the and structures (Fig. A is the of the and In the this is the by to the (Fig. with the of P. aeruginosa, the is in with the the Finally, with the the in the structures is more and more to the of and the these two forms, a reorganization of the of product-bound GmhA together a with and and the the structural and as in in the formation of in the of GmhA is for the to and product are at the A and only one of the within GmhA substrate, whereas the structure of product-bound GmhA In the structure and the acid side and of and and of A. In the for the and product-bound structures are (Fig. residues from structures in and in to product-bound P. aeruginosa not side from residues and not significantly in position the two In residues and to the of position in the The in the product-bound by additional with residues and of (Fig. The that residues from and are in product that of a GmhA be for for GmhA residues for analysis based on data of the E. coli enzyme and product-bound P. aeruginosa enzyme (Fig. A of residues for analysis and is by E. coli residues of and in and product crystal The highly conserved and to the S7P play a role in only one from this for and are unique to the E. coli whereas and are unique to the P. aeruginosa product-bound structure. in residues are conserved Gram-negative with the of to its position in the E. coli In of E. coli GmhA and to synthesized S7P into product using a using that synthesized S7P a of and that the for at in the of of GmhA then for activity against S7P the only and in to that of GmhA whereas the of that of The GmhA mutants, and in activity of at of of and E. coli GmhA GmhA in a new In the role of in the in vivo studies using E. coli and A E. coli and E. coli deletion also of GmhA and therefore to the in by The growth of to the of gmhA not have the first growth of as by the of and as as the a of and to with the to increase in the of a the or absence of in or has on the growth of E. coli The susceptibility of these gmhA to the antibiotic novobiocin then (Fig. with an LPS, E. coli is to In E. coli a only lipid A and Kdo are increased sensitivity to novobiocin S. M. J. Bacteriol. 1971; PubMed Google Scholar). in the with the a novobiocin of with an of for the The and to novobiocin to levels (Fig. The increased sensitivity to within one The as as and in within one that of the The a whereas the represents a in with LPS from of the gmhA the the GmhA forms and using in this LPS be from LPS based on LPS through the in and GmhA not LPS the these The GmhA and in at The to with GmhA the to and GmhA these to be at in The of is also to the novobiocin with on these that LPS is not to sensitize E. coli to LPS be as in the the membrane to a which the permeability barrier is as with the and of have the structures of GmhA from E. coli and the opportunistic bacterial pathogen P. aeruginosa, in apo, and product-bound These structures structures from C. and that have from structural studies J. S. 2006; PubMed Scopus Google Scholar). The E. coli and C. as in the whereas the P. aeruginosa and as as is the the of the do not of studies in this suggest that the of GmhA is a is also by the in formation for and product-bound the structures of is that structures be into of two distinct an and a The is by an an and a In the the region adopts a structure that in not only of the the by but also more interactions resulting in a more The is in structures of GmhA in and forms from E. coli and also in the structures from P. aeruginosa and The is in the and product-bound structures from C. and P. aeruginosa, The that only two conformations are structures been in from organisms, and in of that GmhA is to in two distinct The and conformations represent structures for and GmhA is an isomerase and be to and that S7P and d-glycero-α,β-d-manno-heptose 7-phosphate are of GmhA. this in is not that the structure of C. GmhA in the product with of GmhA of from in vivo and in analysis of GmhA studies suggest the of residues and in enzyme acid at and residues in GmhA activity in using the phosphate activity of these these residues are within the However, to the role of these residues in GmhA action from analysis this in vivo studies in and in vivo studies suggest that and play significant role in GmhA activity and in the LPS permeability barrier as in novobiocin studies and by LPS these residues are to GmhA The and not the permeability of E. coli to not that and have role in are a of these activity in and not in the of studies to the a Furthermore, in which could to activity based on the of in the this in be in as with an of the activity of and could not be or in the of The role of to be in as predicted by to isomerase (6Valvano M.A. Messner P. Kosma P. Microbiology. 2002; 148: 1979-1989Crossref PubMed Scopus (112) Google Scholar). is that the role of could be by in and which are also predicted to function in phosphate The not in the of the this based on in the product-bound and is unique residues within the and is of is from the residues to or product from the analysis of the GmhA and that the E. coli as a in and data also a the E. coli as a in the and of at of GmhA phosphate and to of GmhA in product from studies are therefore with the that product is an mechanism used by GmhA to isomerization that and as the residues for GmhA a mechanism of action of GmhA be by to other known with and as the catalytic residues. residues are in isomerase as a catalytic the through J. J. J.P. J. Mol. Biol. 2006; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the of the isomerase of glucosamine 6-phosphate has been to on and residues S. A. M. Biochem. 2006; PubMed Scopus (17) Google Scholar, A. M. Full Text Full Text PDF PubMed Scopus Google Scholar). enzyme the structural to GmhA J. S. 2006; PubMed Scopus Google Scholar). A structural of the quaternary and structures of these two isomerase enzymes is in a of and residues forms a conserved The catalytic in E. coli GmhA is also for which is replaced by a in the catalytic of has in GmhA. The catalytic of is not conserved to within GmhA. analysis not suggest that GmhA a for as by in the S7P sugar to as a two of these is to a of GmhA. Thus, to the isomerase of adopts an quaternary structure of additional for the of a GmhA A potential mechanism of action for based on studies of the isomerase from is in mechanism that or could act as the catalytic a from of the S7P substrate, the other would act as a catalytic a to for The then through the resulting resulting in an which then to give the d-glycero-α,β-d-manno-heptose is to which or catalytic based on the absence of activity is be that the role of the catalytic in this mechanism. the and studies new into the of an essential in the permeability barrier of Gram-negative bacteria. GmhA is highly conserved pathogenic in sequence and structure. from the studies of GmhA from E. coli and P. aeruginosa be to other pathogenic Gram-negative of in with known antimicrobial aid in treatment of Gram-negative of the structure and mechanism of GmhA are the first in the heptose biosynthetic pathway as a novel Gram-negative antimicrobial We and for with and J. of the for for the selenomethionine P. aeruginosa with

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