antibiotic resistance , one of the most 
 pressing problems in the management of infectious diseases , is causing 
 a crisis in the health - care sector . multidrug resistance ( mdr ) has 
 been rapidly evolving in bacterial species commonly associated with 
 infections in hospitals and communities , as a consequence of vertical 
 gene transfer within each species and horizontal gene transfer among 
 different species . while mdr in gram - positive 
 bacteria ( e.g. , staphylococcus aureus , enterococcus 
 faecium ) has been receiving much attention from the pharmaceutical 
 sector , efforts for overcoming that in gram - negative bacteria ( e.g. , burkholderia cenocepacia , pseudomonas aeruginosa , escherichia coli ) have been limited , despite the 
 fact that many of the latter bacteria are well - known for being among 
 the most common and the most dangerous opportunistic pathogens to 
 immunocompromised individuals such as cystic fibrosis patients and 
 that the efficacy of currently available antibiotics in combating 
 these bacteria has been declining . new therapeutic strategies and 
 new antibiotics are urgently needed in confronting the problem of 
 mdr . a variety of physiological processes 
 in bacteria have been identified as targets for new antibacterial 
 therapeutics ; for example , peptidoglycan biosynthesis , drug efflux , 
 and dna topoisomerization . the defining characteristic of gram - negative 
 bacteria is the presence of an outer membrane , an asymmetric lipid 
 bilayer whose outer leaflet is mainly composed of lipopolysaccharide 
 ( lps ) . lps forms a dense mesh and helps to anchor proteins in the 
 outer membrane . on one hand , this maintains the structural integrity 
 of the outer membrane , which is essential for adhesion to host cells 
 and interaction with host immune effectors including serum components . the dense hydrophilic mesh of lps renders the 
 outer membrane impermeable to hydrophobic molecules , which include 
 many currently available antibiotics such as macrolides . lps comprises lipid a , a core oligosaccharide 
 ( os ) and , in some bacterial species , an o - antigen polysaccharide chain . the core os can be further divided into an inner core region consisting 
 of 2-keto-3-deoxy - d - manno - octosonic acid ( kdo ) and heptose 
 ( prevalently l--d - manno - heptose ) residues 
 and an outer core region consisting mostly of hexose residues ( figure 1a ) . lipid a and kdo are indispensable 
 for cell viability and conserved in virtually all gram - negative bacteria . interestingly , mutant cells with minimal lps ( i.e. , lps comprising 
 lipid a and kdo only ) are viable but display the so - called deep - rough 
 phenotype . in particular , they exhibit increased sensitivity to hydrophobic 
 molecules owing to the lack of negatively charged groups in the truncated 
 lps for cross - linking by divalent cations , which disfavors the formation 
 of a dense mesh in the outer membrane . in most species , these negatively 
 charged groups are contributed by phosphorylated heptose residues . ( b ) biosynthesis of adp - l - glycero--d - manno - heptose , a precursor for the biosynthesis of the inner 
 core region of lps , from d - sedoheptulose 7-phosphate . the 
 phosphorylation reaction converting d - glycero--d - manno - heptose-7-phosphate ( m7p ) to d - glycero--d - manno - heptose-1,7-bisphosphate ( gmb ) is catalyzed by hlda . ic50 values against 
 hlda are the following : 0.81 m for 1 ; 0.23 m 
 for 2 . adp - l - glycero--d - manno - heptose 
 is a precursor for the biosynthesis of the inner core region of lps . the pentose phosphate pathway supplies d - sedoheptulose 7-phosphate , 
 which is converted to adp - l - glycero--d - manno - heptose 
 in five steps . specifically , the second step is a phosphorylation 
 reaction converting d - glycero--d - manno - heptose-7-phosphate 
 ( m7p ) to d - glycero--d - manno - heptose 1,7-bisphosphate 
 ( gmb ) , and the fourth step is a nucleotidyl transfer reaction converting d - glycero--d - manno - heptose 1-phosphate to adp - d - glycero--d - manno - heptose . in most species ( e.g. , e. coli , p. aeruginosa , klebsiella pneumonia ) , a 
 single bifunctional enzyme , hlde , catalyzes these two steps ; but in 
 some others ( e.g. , b. cenocepacia , neisseria 
 meningitidis , neisseria gonorrhoeae ) , two 
 separate enzymes , hlda and hldc , catalyze the second and the fourth 
 steps , respectively ( figure 1b ) . hlda and hldc 
 are homologous to the n- and the c - terminal domains of hlde , respectively . previous studies have shown for some of the 
 adp - l - glycero--d - manno - heptose biosynthetic 
 enzymes that deletion or mutation of their corresponding genes leads 
 to the production of heptoseless lps , resulting in increased bacterial 
 sensitivity to antibiotics and increased susceptibility to lysis mediated 
 by the host complement . it is hypothesized that inhibition 
 of the biosynthesis of adp - l - glycero--d - manno - heptose 
 results in the production of defective lps , thereby increasing the 
 sensitivity of gram - negative bacteria to host defense and hydrophobic 
 antibiotics . absent in human cells , this biosynthetic pathway presents 
 a highly advantageous therapeutic target . inhibitors of adp - l - glycero--d - manno - heptose biosynthetic enzymes can 
 serve as antibiotic adjuvants , coadministered with currently available 
 antibiotics to enhance their efficacies . alternatively , these inhibitors 
 could be administered alone to prevent infections in immunocompromised 
 patients . to facilitate structure - based inhibitor design , crystal 
 structures have been determined previously for some of the adp - l - glycero--d - manno - heptose biosynthetic enzymes . here , we report four crystal structures of hlda , one showing simultaneously 
 its enzyme  substrate and enzyme  product complexes ( es / ep ) 
 and three showing complexes with two potent inhibitors ( 1 and 2 , figure 1c ) , which were 
 designed using medicinal chemistry from a hit identified by high - throughput 
 screening ( that work is reported in the subsequent article , in which 
 inhibitors 1 and 2 are described as compounds 25 and 85 , respectively ) . to our knowledge , these structures are the first ever reported for 
 a heptose kinase , providing valuable insights into the mechanisms 
 of its catalysis and inhibition , which will aid in the development 
 of drugs with antivirulent and membrane - permeabilizing properties . the amino acid sequence of hlda includes the two signature motifs 
 ( the n - terminal gly - rich motif and the c - terminal gxgd motif ) characterizing 
 members of the pfkb carbohydrate kinase family . a search of the protein data bank using the dali server 
 shows that hlda structurally aligns best with members of the pfkb 
 carbohydrate kinase family ( z - scores mostly higher 
 than 20 ) despite low sequence identities ( below 25% ) . the / core adopts 
 a variant rossmann fold , with a twisted nine - stranded -sheet 
 ( 5418910111213 ) 
 sandwiched by five -helices on one face ( 4 , 5 , 
 6 , 7 , and 8 ) and four -helices on the 
 opposite face ( 2 , 3 , 9 , and 10 ) . a short 
 -helix forms at both the n- ( 1 ) and the c - termini ( 11 ) 
 of each protomer . 1 is located near the n - terminus of 4 
 and the c - termini of 2 and 9 , while 11 is shielded 
 from the -sheet in the / core by 2 , 9 , 
 and 10 . the protruding twisted -sheet is composed of 
 four -strands ( 3267 ) , 
 with 2 and 3 formed in the extended loop connecting 
 1 to 2 , and 6 and 7 in that connecting 
 5 to 4 ( figure 2a ) . the two protomers 
 in the asymmetric unit are related by noncrystallographic 2-fold symmetry , 
 interacting with each other through their protruding twisted -sheets . their protruding twisted -sheets are oriented orthogonally 
 with respect to each other , and the 3 of each protomer has 
 its n - terminus bent to form hydrogen bonds with the 7 of the 
 opposite protomer in a parallel -sheet fashion . this leads 
 to the formation of a slightly flattened -barrel with its openings 
 orthogonal to the 2-fold axis ( also known as -clasp , figure 2b ) . a total of 2250 
  of solvent - accessible surface area is buried upon 
 dimerization . importantly , the -guanidinium group of arg38 at the c - terminus 
 of 2 of each protomer forms ionic interactions with the side 
 chain carboxylate groups of asp127 and glu129 at the c - terminus of 
 7 of the opposite protomer , while the -carboxylate group 
 of glu42 in the loop connecting 2 to 3 of each protomer 
 forms ionic interactions with the -guanidinium groups of arg115 
 in 6 and arg125 in 7 of the opposite protomer . superimposition 
 of the two protomers of hlda ( based on main - chain atoms ) yielded an 
 rmsd of 0.26  , indicating that both protomers adopt essentially 
 the same structure and conformation . the n- and the c - termini and the secondary structure elements are 
 labeled . for all of the members of the pfkb carbohydrate 
 kinase family whose three - dimensional structures have been determined 
 so far , the active site is located between the / core 
 and the protruding twisted -sheet of each protomer . interestingly , 
 at the initial stage of structural model refinement , outstanding electron 
 densities were found between the / core and the protruding 
 twisted -sheet in both protomers of hlda . the shapes and the 
 magnitudes of these electron densities strongly indicate that the 
 hydrolyzed atp analogue and the substrate of hlda , amppn and 
 m7p , bind in this location of protomer a ( figure 3a ) , whereas amppn and gmb ( the product of hlda ) bind in this 
 location of protomer b ( figure 3b ) . both m7p 
 and gmb are in the -pyranose form and in the chair conformation . 
 the gxgd signature motif ( gly267-ala268-gly269-asp270 , with asp270 
 being putatively central to the catalytic function ) forms the n - terminus 
 of 9 , located between the nucleotide and the heptose in both 
 protomers . residual electron densities in the ( 2|fo|  |fc|)c map 
 of hlda es / ep contoured at 1.0 : ( a ) active site of protomer 
 a ; ( b ) active site of protomer b. asp270 , amppn , m7p , and gmb 
 are shown with stick models . , the nucleotide - binding site 
 is constituted solely by residues from the / core . the 
 shapes of the outstanding electron densities clearly indicate that 
 the adenine ring of the nucleotide is in the syn orientation , 
 making van der waals contacts with the main - chain atoms of ser240 
 and glu241 in the loop connecting 11 to 12 and with 
 the side chains of ala257 , ala259 , val262 , and val265 in the loop 
 connecting 13 to 9 and val301 at the c - terminus of 10 . the ribose ring of the nucleotide is in the c3-endo conformation , with its 2-hydroxyl group hydrogen - bonded 
 to the -amide group of asn294 in 10 and with its 3-hydroxyl 
 group hydrogen - bonded to the main - chain carbonyl group of gly243 at 
 the n - terminus of 12 . the negative charges of the phosphate 
 groups of the nucleotide are accommodated by the positive ends of 
 the helix dipoles of 7 and 9 , with the n - terminus of 
 7 in a distance of approximately 6  from both the - 
 and the -phosphate groups and with the n - terminus of 9 
 in a distance of approximately 3.5  from the -phosphate 
 group . the -phosphate group forms hydrogen bonds through its 
 oxygen atom o1a with the main - chain nh group of ser240 , through o1a 
 and o2a with the -hydroxyl group of ser240 , and through o1a 
 and o3a with the -hydroxyl group of thr238 at the c - terminus 
 of 11 . the -phosphate group forms hydrogen bonds through 
 o1b with the -amide group of asn202 at the n - terminus of 7 
 and through n3b with the main - chain nh group of gly269 . residues asn202 
 to glu205 constitute the nxxe motif conserved in many members of the 
 pfkb carbohydrate kinase family . the observed interaction suggests 
 that the asparagine of this motif helps to align the phosphate groups 
 of the nucleotide for the phosphorylation reaction . interestingly , 
 o2b of the -phosphate group forms a dative bond with a magnesium 
 ion ( figure 4 ) . the assignment of a magnesium 
 ion instead of a water molecule at this position is strongly supported 
 by the quasi - octahedral coordination geometry exhibited by the side 
 chain carboxylate groups of asp184 at the c - terminus of 9 and 
 glu205 at the n - terminus of 7 , amppn , gmb ( in protomer 
 b ) , and several water molecules in this region . studies on ribokinases 
 and adenosine kinases from different species , which belong to the 
 pfkb carbohydrate kinase family as well , have shown that divalent 
 cations ( presumably a magnesium ion in vivo ) are required for catalysis . moreover , mutagenesis studies on some 
 other members of this family have shown that the glutamate of the 
 nxxe motif , which corresponds to glu205 in hlda , is important for 
 the binding of a magnesium ion in the active site . the -phosphate group of amppnp could not be 
 located in either protomer because of the lack of electron densities . only the site in protomer a is shown , as the nucleotide - binding 
 interactions in protomer b are essentially the same . amppn , 
 m7p , and all of the residues involved are shown with stick models , 
 while the magnesium ion is shown in cyan . for clarity , only some of 
 the residues are labeled . in contrast to the nucleotide - binding site , the 
 heptose - binding site in each protomer is constituted by residues from 
 both the / core and the protruding twisted -sheet 
 and by two residues from the loop connecting 2 to 3 
 of the opposite protomer . importantly , in protomer a , the -carboxylate 
 group of asp270a forms a hydrogen bond with the 1-hydroxyl group of 
 m7p . the -hydroxyl group of tyr159 forms a hydrogen bond with 
 the 1-hydroxyl group of m7p as well in protomer a and with the phosphoester 
 oxygen atom at position 1 of gmb in protomer b. in both protomers , 
 the 2-hydroxyl group of the heptose forms hydrogen bonds with the 
 main - chain nh group of gly59 at the c - terminus of 3 , the -hydroxyl 
 group of tyr159 and the -carboxylate group of asp270 . the 3-hydroxyl 
 group of the heptose forms hydrogen bonds with the -carboxylate 
 group of asp29 in 2 and the main - chain nh group of gly59 , while 
 the 4-hydroxyl group of the heptose forms hydrogen bonds with the 
 -carboxylate group of asp29 and the -guanidinium group 
 of arg115 , the latter of which is positioned by the -carboxylate 
 group of glu42 of the opposite protomer through an ionic interaction . the 6-hydroxyl group of the heptose forms hydrogen bonds with the 
 -guanidinium 
 group of arg125 , which is positioned by the side chain carboxylate 
 groups of asp127 of the parent protomer and glu42 of the opposite 
 protomer through ionic interactions . importantly , the -guanidinium 
 group of arg125 , together with the -amine groups of lys113 
 at the n - terminus of 6 , lys161 in the loop connecting 8 
 to 5 , and lys186 in the loop connecting 9 to 6 
 of the parent protomer , and with the -guanidinium group of 
 arg38 of the opposite protomer , also forms ionic interactions with 
 the 7-phosphate group of the heptose ( figure 5 ) . this partly accounts for the specificity of hlda for m7p . through 
 ionic interactions , the -amine group of lys113 is positioned 
 by the side chain carboxylate groups of asp127 and glu129 ; the -amine 
 group of lys161 is positioned by the -carboxylate group of 
 glu129 , and the -amine group of lys186 is positioned by the 
 side chain carboxylate groups of asp184 and glu205 . in protomer b , 
 the 1-phosphate group of gmb was assigned half occupancy during structural 
 model refinement because its less well - defined electron densities 
 suggest that only a limited amount of m7p had been phosphorylated . no significant structural or conformational differences were observed 
 between the two protomers that could account for the binding of m7p 
 in protomer a and gmb in protomer b. examination of crystal packing 
 did not reveal any relevance with this mode of binding either . only the site in protomer a is shown , as the heptose - binding 
 interactions in protomer b are essentially the same . amppn , 
 m7p , and all of the residues involved are shown with stick models , 
 while the magnesium ion is shown in cyan . interestingly , in protomer a , two water molecules 
 were located between the -phosphate group of amppn and 
 the 1-hydroxyl group of m7p . it is possible that the electron densities 
 of these two water molecules actually belong to two of the oxygen 
 atoms of the dynamically disordered -phosphate group of amppnp . 
 on the basis of the positions of these two water molecules , the -phosphate 
 group was modeled . the phosphorus atom of the -phosphate group 
 of amppnp and the oxygen atom of the 1-hydroxyl group of m7p 
 are aligned head - on at a distance of 3.1  ( figure 6a ) . in protomer b , the 1-phosphate group of gmb forms hydrogen 
 bonds through op1 with the main - chain nh groups of gly269b and asp270b 
 and through op2 with the main - chain nh group of gly267 . the distance between n3b of 
 the -phosphate group of amppn and the phosphorus atom 
 of the 1-phosphate group of gmb is 4.8  ( figure 6b ) . these observations together strongly indicate the feasibility 
 of an in - line mechanism , which has been proposed for other members 
 of the pfkb carbohydrate kinase family , being adopted by the hlda - catalyzed 
 phosphorylation . asp270 of the gxgd signature motif acts as a catalytic 
 base , with its -carboxylate group deprotonating the 1-hydroxyl 
 group of m7p . the deprotonatation of the 1-hydroxyl group of m7p favors 
 its oxygen atom performing a nucleophilic attack at the phosphorus 
 atom of the -phosphate group of atp . the gxgd signature motif 
 forms an anion hole at the n - terminus of 9 , in which the positive 
 end of the helix dipole of 9 and the hydrogen bonds donated 
 by the main - chain nh groups of the gxgd signature motif , together 
 with the magnesium ion , help to accommodate the additional negative 
 charge in the -phosphate group of atp during the nucleophilic 
 attack , thereby lowering the energy of the transition state . the nucleophilic 
 attack results in the formation of a phosphoester bond between the 
 1-hydroxyl group of m7p and the -phosphate group of atp and 
 in the dissociation of the phosphodiester bond between the - 
 and the -phosphate groups of atp , thereby producing gmb and 
 adp ( figure 6c ) . catalytic mechanism of 
 hlda . ( a ) on the basis of the positions of the two water molecules 
 ( shown in red and labeled w ) located between amppn and m7p ( both 
 shown with stick models ) in protomer a , an additional phosphate group 
 ( circled in red ) was modeled onto amppn . the head - on alignment 
 of the -phosphate group of amppnp with the 1-hydroxyl 
 group of m7p is indicated by a dotted line . residues of the gxgd signature 
 motif are shown with stick models as well , while the magnesium ion 
 is shown in cyan . ( b ) in protomer b , the 1-phosphate group of gmb 
 forms hydrogen bonds with the main - chain nh groups of residues of 
 the gxgd signature motif and an ionic interaction with the magnesium 
 ion ( all indicated by dashed lines ) . a plasmid encoding the wild - type hlda ( psl3 ) has been tested previously 
 for its ability to complement the lps phenotype of a b. cenocepacia hlda mutant strain ( rsf39 ) . upon gel electrophoresis 
 and silver staining , lps extracted from the parental strain exhibited 
 a ladder - like appearance , indicating the presence of lipid a , complete 
 core os , and an o - antigen polysaccharide chain with a variable number 
 of saccharide units . rsf39 conjugated with the vector control pscrhab2 
 produced lps with a truncated ( heptoseless ) core os , which migrated 
 as a single band of low molecular weight . in contrast , psl3 restored 
 in rsf39 the lps phenotype of the parental strain . on the basis of the structure of hlda es / ep , seven residues 
 were selected for an assessment of their importance in the in vivo 
 activity of hlda using this method . a plasmid encoding hlda y159f 
 restored the parental lps phenotype in rsf39 , whereas one encoding 
 hlda d270a showed an lps profile similar to that of rsf39 carrying 
 the vector control pscrhab2 . these data support the conclusion that 
 the catalytic residue of hlda is asp270 , not tyr159 . the heptose - binding 
 site of each protomer involves glu42 of the opposite protomer . a plasmid 
 encoding the e42a variant partially restored the parental lps phenotype 
 in rsf39 , indicating that glu42 is not absolutely required for the 
 in vivo activity of hlda . this is consistent with the apparent lack 
 of direct participation of glu42 in the catalytic mechanism of hlda . however , a significant amount of lps extracted from the conjugated 
 rsf39 consisted of lipid a and a truncated core os , indicating that 
 glu42 is a significant contributor to the in vivo activity of hlda . 
 this can be explained by the importance of glu42 in the formation 
 of a catalytically favorable heptose - binding site : glu42 of each protomer 
 positions arg115 and arg125 of the opposite protomer for interacting 
 with the 4-hydroxyl , the 6-hydroxyl , and the 7-phosphate groups of 
 m7p . results of the mutagenesis studies did not reflect the importance 
 of asp184 , lys186 , asn202 , and glu205 suggested by the structure of 
 hlda es / ep . plasmids encoding the d184a , the k186a , the n202a , and 
 the e205a variants restored the parental lps phenotype in rsf39 similarly 
 as psl3 did . possibly , the functional effects of these substitutions 
 are not significant in vivo or are diminished by other residues in 
 proximity ( figure 7 ) . lps profiles . lps extracted 
 from the wild - type b. cenocepacia strain k56 - 2 and 
 from the k56 - 2 hlda mutant ( rsf39 ) conjugated 
 with the vector control pscrhab2 , with plasmids encoding substitutions 
 of the seven selected residues in hlda ( psl3 site - directed mutants ) , 
 and with a plasmid encoding the wild - type hlda ( psl3 ) was separated 
 by gel electrophoresis and stained with silver nitrate . bands with 
 ladder - like appearance near the top of the gel correspond to lps molecules 
 with lipid a , a complete core os , and an o - antigen polysaccharide 
 chain with a variable number of saccharide units . near the bottom 
 of the gel , the band of higher molecular weight ( as seen for k56 - 2 ) 
 corresponds to lps molecules with lipid a and a complete core os , 
 whereas the band of lower molecular weight ( as seen for rsf39 ) corresponds 
 to lps molecules with lipid a and a truncated ( heptoseless ) core os . we have identified 
 quercetin , piceatannol , and several compounds in the tyrphostin family 
 as hlda inhibitors in a previous screen ( unpublished results ) . these 
 compounds are broad - spectrum kinase inhibitors lacking the selectivity 
 required for serving as leads in drug development . inhibitors 1 and 2 were optimized using medicinal chemistry 
 from a high - throughput screening hit discovered at mutabilis against 
 the n - terminal kinase domain of e.coli hlde ; that 
 work is reported in the subsequent article , in which inhibitors 1 and 2 are described as compounds 25 and 85 , respectively . inhibitors 1 and 2 were shown to inhibit b. cenocepacia hlda at ic50 values of 0.81 and 0.23 m , respectively . the overall structure of hlda is preserved in all of the three 
 hlda  inhibitor complexes . at the initial stage of structural 
 model refinement , outstanding electron densities were found in the 
 nucleotide - binding site of each protomer of these complexes . the shapes 
 and the magnitudes of these electron densities indicate that inhibitor 1 binds in this location of each protomer of his6-hlda:1 and hlda : m7p:1 ( figure 8) while inhibitor 2 binds in this location 
 of each protomer of hlda:2 . outstanding electron densities 
 were found in the heptose - binding site of each protomer of hlda : m7p:1 as well . those in protomer a were of sufficient quality 
 to justify the assignment of m7p in the -pyranose form and 
 in the chair conformation ( figure 8) , whereas 
 those in protomer b could justify the assignment of only a phosphate 
 ion that corresponds to the 7-phosphate group of m7p . residual electron densities 
 in the ( 2|fo|  |fc|)c maps of hlda : m7p:1 ( protomer a ) contoured at 1.0. electron densities associated 
 with the inhibitors were observed in protomer b of hlda : m7p:1 and both protomers of his6-hlda:1 and hlda:2 as well . both inhibitors 1 and 2 improve their shape complementarity with the nucleotide - binding 
 sites of hlda by adopting a folded conformation , with their phenyltriazine , 
 linker , and benzothiazole moieties roughly corresponding to the adenine , 
 the ribose , and the phosphate moieties of amppn , respectively 
 ( figure 9a ) . however , compared with amppn , 
 these inhibitors penetrate the nucleotide - binding sites more deeply , 
 thereby enhancing their hydrophobic interactions with the protein , 
 which constitute the majority of hlda  inhibitor interactions 
 ( figure 9b ) . these inhibitors make van der 
 waals contacts with the main - chain atoms of ser240 , glu241 , gly243 , 
 gly269 , and gly298 and with the side chains of asn202 , thr238 , met244 , 
 ala257 , ala259 , val262 , val265 , ala268 , val272 , ala297 , and val301 . n3 of the benzothiazole moiety forms a hydrogen bond with the 
 main - chain nh group of ser240 , while the nh group of the linker moiety 
 forms a hydrogen bond with the -amide group of asn294 . inhibitor 1 has a methoxyl substituent 
 at phenyl c2 and c6 , whereas inhibitor 2 has a chloride 
 substituent at the corresponding positions . nevertheless , no significant 
 differences were observed between these inhibitors in their interactions 
 with hlda . for both inhibitors , one of the substituents protrudes 
 into the solvent , whereas the other interacts with the benzothiazole 
 moiety in a lipophilic ( for methoxyl ) or a chloride 
 ( for chloride ) manner ( figure 9a ) . importantly , 
 in protomer a of hlda : m7p:1 , no interactions were observed 
 between inhibitor 1 and m7p , indicating that the binding 
 of either ligand does not favor or disfavor that of the other ( figure 10 ) . this strongly suggests that inhibitors 1 and 2 do not use the exclusion of m7p as a 
 strategy for inhibiting hlda . comparisons 
 among the inhibitors and amppn in 
 the nucleotide - binding site of hlda . ( a ) inhibitor 1 ( shown 
 with pink and white stick models , as observed in protomer b of his6-hlda:1 and protomer a of hlda : m7p:1 , respectively ) versus inhibitor 2 ( shown with a cyan 
 stick model , as observed in protomer a of hlda:2 ) . ( b ) 
 inhibitor 1 ( shown with a white stick model , as observed 
 in protomer a of hlda : m7p:1 ) versus amppn ( shown 
 with a gold stick model , as observed in protomer a of hlda es / ep ) . the protein surface was calculated and m7p was shown based on the 
 structure of hlda : m7p:1 . only the nucleotide - binding 
 site in protomer a of hlda : m7p:1 is shown , as the inhibitor - binding 
 interactions are essentially the same in protomer b of hlda : m7p:1 and both protomers of his6-hlda:1 and hlda:2 . inhibitor 1 , m7p , and all 
 of the residues involved are shown with stick models . inhibitor 
 binding on hydrophobic interactions suggests that the potential of 
 hydrophobic interactions should be fully exploited in the design of 
 new hlda inhibitors . the shape complementarity 
 could be further improved by adding substituents to some positions 
 of the inhibitor , provided that these substituents would not sterically 
 hinder the folding of the inhibitor or prevent its entry into the 
 nucleotide - binding sites of hlda . on the other hand , the potential 
 of hydrogen - bonding and ionic interactions , the linker and the benzothiazole moieties of the inhibitor 
 could be redesigned to interact with residues of hlda that form hydrogen 
 bonds with amppn in hlda es / ep . hlda functions as a homodimeric enzyme in which 
 each protomer comprises an / core providing the platform 
 for catalytic activity and a protruding twisted -sheet mainly 
 involved in dimerization . the nucleotide - binding interactions and 
 the heptose - binding interactions of hlda share similar properties 
 with their respective counterparts in other proteins . the adenine 
 ring of the nucleotide forms hydrophobic interactions with hlda , while 
 the ribose ring and the phosphate groups of the nucleotide interact 
 with hlda mainly through hydrogen bonding . in addition , several positively 
 charged residues , which are positioned by several negatively charged 
 residues of the same and the opposite protomers through a network 
 of ionic interactions , contribute to the specificity of hlda for m7p . 
 apparently , the hlda - catalyzed phosphorylation adopts the in - line 
 mechanism proposed for other members of the pfkb carbohydrate kinase 
 family as well , in which the transition state is stabilized by a magnesium 
 ion , the anion hole ( formed by the gxgd signature motif ) , and the 
 helix dipole of 9 of hlda . results of the mutagenesis studies 
 on the in vivo activity of hlda confirm the role of the conserved 
 aspartate , asp270 , as the catalytic residue . both inhibitors 1 and 2 bind to the nucleotide - binding sites 
 of hlda without altering the overall structure of the enzyme or interfering 
 with the binding of m7p . both inhibitors adopt a folded conformation 
 to improve their shape complementarity with the nucleotide - binding 
 sites of hlda and penetrate the sites more deeply than amppn 
 does to enhance their hydrophobic interactions with the enzyme . the 
 hlda  inhibitor interactions are mainly hydrophobic in nature 
 with limited contribution by hydrogen bonding and dipole  dipole 
 interaction . the structures of the hlda  inhibitor complexes 
 provide clues for rational modification of the inhibitors . urged 
 by the rapid evolution of mdr , research in bacterial physiology has 
 been very active in identifying targets for new antibacterial therapeutics . interest has been growing in lps biosynthesis 
 as a target for the development of antivirulence drugs and antibiotic 
 adjuvants against gram - negative bacteria . in particular , the biosynthetic 
 pathway of adp - l - glycero--d - manno - heptose , 
 the precursor for lps biosynthesis , has recently been gaining attention 
 from the pharmaceutical sector . this work provides essential structural  functional information 
 of hlda , an important enzyme in the biosynthetic pathway of adp - l - glycero--d - manno - heptose , and valuable insight 
 into its catalytic mechanism , thereby supporting medicinal chemists 
 in their design of new hlda inhibitors . in view of the general concern 
 about the lack of selectivity of inhibitors targeting nucleotide - binding 
 sites , it is expected that future attention will be drawn to the pursuit 
 of inhibitors targeting the heptose - binding sites of hlda . design 
 of bivalent inhibitors targeting both the nucleotide- and the heptose - binding 
 sites of hlda presents an exciting prospect in the long run as well . furthermore , most gram - negative bacterial species employ hlde instead 
 of hlda and hldc in the biosynthesis of adp - l - glycero--d - manno - heptose . however , studies of hlde have been hindered 
 by the lack of structural information . crystallization of hlde has 
 been a major challenge probably because of the dynamic behaviors of 
 this two - domain protein . because of the homology of hlda with the 
 n - terminal domain of hlde , this work will be of great importance for 
 our understanding of hlde and the design of new hlde inhibitors as 
 well . all chemicals , unless otherwise specified , 
 were purchased from various commercial sources and used without further 
 purification . the synthesis of inhibitors 1 and 2 is reported in the supporting information of the subsequent 
 article ( therein described as compounds 25 and 85 , respectively ) . both inhibitors 
 display hplc purity of > 95% and have been characterized by h nmr , ms , and crystallographic data . b. cenocepacia 
 hlda gene was cloned into the gateway pdest17 vector using 
 the gateway cloning system ( invitrogen ) . chemically competent cells 
 of the e. coli strain bl21(de3)-t1 ( sigma ) 
 were transformed with pdest17-hlda , liquid - cultured 
 to an optical density of 0.5 at a wavelength of 600 nm , and induced 
 with 1 mm isopropyl -d-1-thiogalactopyranoside for 
 4 h. cells were harvested by centrifugation at 2392 g for 20 min and resuspended in the lysis buffer ( 50 mm tris , ph 8.0 , 
 40 mm imidazole , 2.8 mm 2-mercaptoethanol , and 500 mm potassium chloride ) 
 with peptidase inhibitors added ( 2 m leupeptin , 2 m 
 pepstatin a , and 2 mm benzamidine hydrochloride ) . resuspended cells 
 were lysed using the french press method at a pressure of 1000 psi 
 for 4 times . the supernatant was filtered with pall metricel membranes 
 with a pore size of 0.45 m and loaded onto a hitrap chelating 
 hp column ( ge healthcare ) precharged with 100 mm nickel chloride and 
 pre - equilibrated with the lysis buffer . the column was washed with 
 30 mm imidazole in the lysis buffer , and elution was with 600 mm imidazole 
 in the lysis buffer , using the kta fast protein liquid chromatography 
 system ( ge healthcare ) . the eluate was passed through a hiprep 26/10 
 desalting column ( ge healthcare ) pre - equilibrated with either the 
 crystallization buffer ( 10 mm tris , ph 7.5 , 170 mm potassium chloride , 
 and 1 mm edta , if his6-hlda was the desired final product ) 
 or the proteolysis buffer ( 50 mm tris , ph 8.0 , 100 mm potassium chloride , 
 and 500 m edta , if hlda was the desired final product ) . to 
 yield hlda , the hexahistidine affinity tag of his6-hlda 
 was removed by incubation at 25 c for 3 h , with 200 g 
 of tobacco etch virus peptidase added . the released hexahistidine 
 affinity tag and any other impurities were eliminated by passing the 
 reaction mixture through the desalting and the chelating columns , 
 both pre - equilibrated with the lysis buffer . the flow - through was 
 passed through the desalting column pre - equilibrated with the crystallization 
 buffer . all of the crystals were grown 
 by the hanging - drop vapor diffusion method . crystals of hlda es / ep 
 were grown at 20 c for several months . the drop contained 1 
 l of the protein solution ( 0.7 mg / ml hlda , 2.5 mm tris , ph 
 7.5 , 42.5 mm potassium chloride , 0.25 mm edta , 1 mm m7p , 1 mm amppnp , 
 5 mm magnesium chloride , and 30% 1,6-hexanediol ) and 1 l of 
 the precipitant solution ( condition no . 27 of hampton research crystal 
 screen , 0.2 m sodium citrate , 0.1 m hepes , ph 7.5 , and 20% v / v 2-propanol ) , 
 while the reservoir contained 500 l of 1.4 m ammonium sulfate . the drop contained 1 l of the protein solution 
 ( 5.0 mg / ml his6-hlda , 10 mm tris , ph 7.5 , 170 mm potassium 
 chloride , 1 mm edta , 150 m m7p , and 500 m inhibitor 1 ) and 1 l of the precipitant solution ( 50 mm hepes , 
 ph 7.5 , 0.05% w / v polyethylene glycol , mr = 400 , and 0.5 m ammonium sulfate ) , while the reservoir contained 
 500 l of 1 m ammonium sulfate . crystals of hlda : m7p:1 were grown at 4 c for 1 month . the drop contained 1 l 
 of the protein solution ( 5.0 mg / ml hlda , 10 mm tris , ph 7.5 , 170 mm 
 potassium chloride , 1 mm edta , 150 m m7p , and 500 m 
 inhibitor 1 ) and 1 l of the precipitant solution 
 ( 0.1 m hepes , ph 7.5 , 10% w / v polyethylene glycol , mr = 10  000 , and 4% v / v ethylene glycol ) , while 
 the reservoir contained 500 l of 1 m ammonium sulfate . crystals 
 of hlda:2 were grown in the same conditions as those 
 of his6-hlda:1 except that hlda and inhibitor 2 were used instead of his6-hlda and inhibitor 1 , respectively . for cryoprotection , all of the crystals were 
 kept at 4 c with the reservoir changed to 500 l of 2 
 m ammonium sulfate for several days before flash - cooling . all of the 
 x - ray diffraction data were collected at the national synchrotron 
 light source at brookhaven national laboratory . all of the data sets 
 were indexed , scaled , and merged using hkl-2000 . initial phases were obtained for hlda es / ep by the single - wavelength 
 anomalous dispersion method , and for his6-hlda:1 , hlda : m7p:1 , and hlda:2 they were 
 obtained by the molecular replacement method using the structure of 
 hlda es / ep as the search model . structural models were built and refined 
 using phenix and were manually adjusted when needed during refinement 
 using coot . the stereochemical quality , the 
 topology , and the interactions ( protein  protein , protein ligand , 
 and protein  metal ion ) within each structural model were analyzed 
 using pdbsum . the cloning vector pscrhab2 , a 
 plasmid encoding the wild - type hlda ( psl3 ) , and psl3 derivatives encoding 
 substitutions of the seven selected residues were transferred to a b. cenocepacia strain k56 - 2 hlda mutant ( rsf39 ) by triparental conjugation . exoconjugants were selected for on lb - agar 
 plates containing 100 g / ml trimethoprim and 50 g / ml 
 gentamicin . exoconjugants were 
 grown overnight on lb - agar plates containing 100 g / ml trimethoprim , 
 with gene expression induced by 0.2% rhamnose . lps was extracted from 
 the cells , separated by gel electrophoresis , and stained with silver 
 nitrate as previously described . the assay 
 buffer ( ab ) consisted of 50 mm hepes , ph 7.5 , 1 mm manganese(ii ) chloride , 
 25 mm potassium chloride , 0.012% triton - x100 , 0.1 m myelin 
 basic protein , and 1 mm dtt . the inhibitor being examined was dissolved 
 at different concentrations in dmso / water or dmso only . then 3 l 
 of each inhibitor solution and 28 l of hlda in ab were mixed 
 in each well of a white polystyrene costar plate . after preincubation 
 at room temperature for 30 min , 29 l of the substrate mixture 
 ( atp and m7p in ab ) was added to each well to give a total volume 
 of 60 l . the reaction mixtures had 50 nm hlda , 0.2 m 
 m7p , 0.2 m atp , and different concentrations of the inhibitor 
 being examined in ab . after incubation at room temperature for 30 
 min , an amount of 200 l of the revelation mixture ( luciferase , d - luciferin and n - acetylcysteamine ) was added 
 to give a total volume of 260 l containing 30 nm luciferase , 
 30 m d - luciferin , and 100 m n - acetylcysteamine . values of percentage inhibition were fitted 
 to a classical equilibrium model with the hill coefficient , nh , using xlfit ( idbs ) :
as an essential constituent of the outer membrane of 
 gram - negative bacteria , lipopolysaccharide contributes significantly 
 to virulence and antibiotic resistance . 
 the lipopolysaccharide biosynthetic 
 pathway therefore serves as a promising therapeutic target for antivirulence 
 drugs and antibiotic adjuvants . here 
 we report the structural  functional 
 studies of d - glycero--d - manno - heptose 7-phosphate 
 kinase ( hlda ) , an absolutely conserved enzyme in this pathway , from burkholderia cenocepacia . 
 hlda is structurally similar to 
 members of the pfkb carbohydrate kinase family and appears to catalyze 
 heptose phosphorylation via an in - line mechanism mediated mainly by 
 a conserved aspartate , asp270 . 
 moreover , we report the structures 
 of hlda in complex with two potent inhibitors in which both inhibitors 
 adopt a folded conformation and occupy the nucleotide - binding sites . 
 together , 
 these results provide important insight into the mechanism 
 of hlda - catalyzed heptose phosphorylation and necessary information 
 for further development of hlda inhibitors .