Cloning and characterization of the Escherichia coli Heptosyltransferase III: Exploring substrate specificity in lipopolysaccharide core biosynthesis

FEBS Letters 589 (2015) 1423–1429

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Cloning and characterization of the Escherichia coli Heptosyltransferase III: Exploring substrate specificity in lipopolysaccharide core biosynthesis Jagadesh Mudapaka, Erika Anne Taylor ⇑ Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA

a r t i c l e

i n f o

Article history: Received 17 March 2015 Revised 22 April 2015 Accepted 23 April 2015 Available online 7 May 2015 Edited by Renee Tsolis Keywords: Lipopolysaccharide biosynthesis Glycosyltransferase Core oligosaccharide Bacterial biofilms Heptosyltransferase

a b s t r a c t Bacterial lipopolysaccharide (LPS) molecules are an important cell surface component that enables adhesion to surfaces and cell motility, amongst other functions. In Escherichia coli, there are multiple Heptosyltransferase enzymes involved in the biosynthesis of the core region of LPS. Here we describe the first ever cloning, expression, purification and characterization of Heptosyltransferase III (HepIII) from E. coli, which catalyzes the addition of an L-glycero-D-manno-heptose (Hep) residue to the growing LPS core via an a(1 ? 7) bond. Inspired by results from our lab on the E. coli HepI, we assessed the catalytic efficiency with phospho-Hep2-Kdo2-Lipid A (PH2K2LA) and two deacylated analogues. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Lipopolysaccharide (LPS), an abundant biomolecule that is a major cell surface component of Gram-negative bacteria, is also an important endotoxin that is recognized for its potential as a glycoconjugate or adjuvant for vaccines [1,2]. Multiple labs have explored the enzymes involved in LPS biosynthesis, including our own which has focused on the Heptosyltransferases that are involved in the biosynthesis of the inner core region of LPS [3–7]. Our studies of Heptosyltransferase I (HepI) from Escherichia coli have revealed that it is the first enzyme in the biosynthetic pathway that does not require lipid functionalization of the Lipid A Abbreviations:

LPS,

lipopolysaccharide; GTs, glycosyltransferases; Hep, HepI, Heptosyltransferase I; HepIII, Heptosyltransferase III; ADP-Hep, adenosine diphosphate-L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; GFP, green fluorescent protein; PH2K2LA, phospho-Hep2-Kdo2-Lipid A; ODH2LA, O-deacylated phospho-Hep2-Kdo2-Lipid A; FDH2LA, fully deacylated phospho-Hep2-Kdo2-Lipid A

L-glycero-D-manno-heptose;

Author contributions: E.T. designed research; J.M. performed research; E.T. and J.M. analyzed data; E.T. and J.M. wrote the paper. ⇑ Corresponding author at: Department of Chemistry, College of the Environment, Wesleyan University, 52 Lawn Ave., Hall-Atwater Labs Room 142, Middletown, CT 06459, USA. Fax: +1 860 685 2211. E-mail address: [email protected] (E.A. Taylor).

based substrate for catalysis [8]. The functional assignment of Heptosyltransferase III (HepIII) from E. coli was performed by Whitfield et al. [7], where they determined through gene disruption studies that HepIII (also known as RfaQ or WaaQ) catalyzes the formation of an a(1 ? 7) glycosidic linkage when it transfers a L-glycero-D-manno-heptose (Hep) to the second Hep of Hep2-Kdo2-Lipid A after phosphorylation by WaaP (Fig. 1). In an effort to better understand the similarities and differences of the Heptosyltransferase enzymes, we herein report the first ever cloning and kinetic characterization of HepIII from E. coli. The substrate scope was explored by assessing catalytic competency of HepIII with a series of Lipid A analogues. Analyses of Heptosyltransferase sequence alignments and a newly developed HepIII structural model were used to inform these findings. This work helps shed light on the similarities and differences of the Heptosyltransferases involved in the LPS inner core biosynthesis, with potential impact on our understanding of glycosyltransferases and beyond.

2. Materials and methods All chemicals were purchased at highest purity from Sigma– Aldrich (St. Louis, MO), unless otherwise indicated.

http://dx.doi.org/10.1016/j.febslet.2015.04.051 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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J. Mudapaka, E.A. Taylor / FEBS Letters 589 (2015) 1423–1429 OH HO HO HO HO HO HO

OH OH O

Hep-II O HO O HO P O O OH HO HO

OH O

NH2

OH

OH OH O

HO HO HO

Hep-I

HO

O

N O O O OO P P O O

N

HO

O

O OH O

HO HO HO

N N

Hep-II

O

OH

O

HO

OH

O HO

O

O

O

HO O

O O P O HO OH O O O

10 12 O

O

10

OH O

OH O

Hep-I O

O OH

O HO

O

O

O O

O

OH

O HO O HO P O O

OH OH

O

Hep-III

O O

NH HO O O HO 10

O

NH

O OH P O

NH2 N

O 10 O

O 10 14

O O O OO P P O O

N O

O O O P O HO O O O

N N

O

O

O O

O

10 12 O

OH OH

O

O O

NH HO O O HO 10

O

NH

O OH P O

O 10 O

O 10

Fig. 1. The overall reaction catalyzed by Heptosyltransferase III from E. coli, which involves the transfer of L-glycero-D-manno-heptose from ADP-Hep to the C7 hydroxyl group of the second heptose of the inner core region of a growing lipopolysaccharide molecule to make an a(1 ? 7) linkage.

2.1. Cloning and expression of HepIII as an N-terminal GFP fusion The E. coli (strain K12) WaaQ gene was PCR amplified from genomic DNA and inserted into the plasmid pNGFP-BC (courtesy of Dr. Eric Gouaux of the Vollum Institute of the Oregon Health & Science University) using the SalI and BamHI restriction sites using an In-Fusion Cloning Kit (Clontech Laboratories, Montain View, CA). The infusion reaction product (N-GFP-HepIII) was transformed into E. coli DH5a cells, grown overnight at 37 °C in LB Gentamycin (12 mg/L) and Ampicillin (100 mg/L), and the gene sequence in the resultant plasmid was confirmed by DNA Analysis Facility at Yale University (New Haven, CT). Each time prior to protein expression, the pNgfp-HepIII vector was extracted using Zyppy™ plasmid mini prep kit (Zymo Research, Irvine, CA), and the plasmid was transferred in to E. coli ArcticExpress cells (Agilent Technologies, Santa Clara, CA) via heat shock using, 5 lL of extracted plasmid DNA and 75 lL of ArcticExpress cells. After allowing the cells to recover in 200 lL of SOC media for one hour at 37 °C, the whole culture was then transferred to a falcon tube containing 20 mL of LB Gentamycin (12 mg/L) and Ampicillin (100 mg/L), and grown overnight with shaking at 37 °C. The overnight culture was used to inoculate multiple flasks of LB-Amp media, which were induced with Isopropyl b-D-1-thiogalactopyranoside (IPTG; Gold Biotechnology, St. Louis, MO) when the OD600 approached 0.6 AU, to give a final concentration of 1 mM IPTG. Growth was then continued at 10 °C for 24 h. The cells were pelleted by centrifugation at 5000 rpm, resuspended in 25 mL/Lcell cultures of Binding buffer (20 mM HEPES, 1 lM imidazole, 0.5 M NaCl, pH 7.5) containing one cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, IN) and lysed using an Emulsiflex™-C5 high pressure homogenizer (Avestin Ottowa, ON) at 15 000 psi for three rounds. Cell debris was removed by centrifugation at 12 000 rpm for 30 min and the supernatant was loaded onto a 5 mL Toyopearl AF Chelate 650 column pre-charged with Cobalt. The column was then washed with 2 column volumes each of binding buffer and wash buffer (20 mM HEPES, 40 mM imidazole, 0.5 M NaCl, pH 7.5). The Cobalt was then removed using 4 column volumes of strip buffer (0.2 M EDTA, 1 M NaCl, and pH 6.8). The fractions containing pure protein were confirmed by SDS–PAGE gels. Fractions with HepIII are combined (typically the late wash and strip fractions), buffer exchanged into the Hep assay buffer (50 mM HEPES,

50 mM KCl, 10 mM MgCl2, pH 7.5) and concentrated using a 30 KMWCO Vivaspin 20 (Vivaproducts, Littleton, MA; also available from GE Healthcare Bio-Sciences, Pittsburgh, PA). The final concentration after buffer exchange of the HepIII was determined using a Bradford assay (standardized against BSA according to kit protocols) and stored at 4 °C in the refrigerator for up to three weeks (efforts to store this enzyme in glycerol containing solutions at lower temperatures, or as an ammonium sulfate precipitate failed to support the maintenance of activity). HepIII loses activity and aggregates slowly over time, necessitating the fresh preparation of enzyme prior to each set of experiments. 2.2. Synthesis of HepIII substrates and analogues The E. coli HepIII (WaaQ) deletion strain, CWG297, was generously provided by Chris Whitfield (University of Guelph). The HepIII knockout cells were grown in 1 L of LB media containing, 10 lg/mL gentamicin at 37 °C overnight, and pelleted by centrifugation at 5000 rpm for 10 min. The phospho-Hep2-Kdo2-Lipid A (PH2K2LA) was then extracted and deacylated as previously described [8–10]. Deacylation reaction mixtures were acidified to pH 5.5, washed twice using dichloromethane and the aqueous layer was loaded onto a G-25 size exclusion column (GE) to separate the O-deacylated and fully deacylated phospho-Hep2-Kdo2-Lipid A (subsequently called ODH2LA and FDH2LA, respectively) from any side products. Products were analyzed on a Finnigan LCQ Advantage Max ESI-MS (Thermo) as previously described [8]. LiODH2LA has a molecular weight of 1858.66 Da and an observed (m/z)2 = 929.46 (Fig. S1a) and NaKFDH2LA has a molecular weight of 1461.21 Da and an observed (m/z)3 = 487.4 (Fig. S1b). ADP-L-glycero-D-manno-heptose (ADP-Hep) was isolated from E. coli WBB06 cells (courtesy of Prof. Christian R.H. Raetz, Duke University), as previously described [8,11,12], (m/z)1 = 618.05 (Fig. S2). 2.3. HepIII kinetic characterization The HepIII enzymatic activity with the above substrates was determined using a CARY 100 Bio UV–vis spectrophotometer (Agilent technologies) to monitor an ADP/NADH coupled assay at 37 °C, analogous to that used for HepI [4,8,13]. The change in the

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absorbance at 340 nm indicates the disappearance of the NADH in response to the conversion of ADP and PEP to ATP and pyruvate by pyruvate kinase (PK), followed by the conversion of pyruvate and NADH to lactate and NAD+ by lactate dehydrogenase (LDH). The assay was performed in buffer containing 50 mM HEPES, 50 mM KCl, 10 mM MgCl2 and pH 7.5, along with 0.01 units each LDH and PK, 100 lM NADH, 100 lM PEP. Using a constant concentration of ADP-Hep (100 lM which was predicted to be well in excess of the Km for APD-Hep based upon the data from HepI and the affinity of HepIII for its acceptor substrate), and variable concentrations of the sugar acceptor substrates (200 nM to 100 lM), kinetics were determined for PH2K2LA, ODH2LA and FDH2LA. Assays were also performed for PH2K2LA and ODH2LA in the presence of 0.1% Triton X-100 detergent to discern the impact on lipidated substrates. For all the assays, the reactions were started by addition of HepIII and mixed well using a micropipette after observing a steady baseline for at least 1 min. The rate data was fitted with KaleidaGraph to the Michaelis–Menten equation for a triplicate set of data. 2.4. HepIII structural model generation Structural models were generated using both Phyre2 [14] and I-TASSER [15] servers. The resulting structural models (all five from I-TASSER and four models generated from Phyre2 with 100.0% confidence ratings) were then compared with both the HepIADP-2-d eoxy-2-fluoroheptose (pdb id = 2H1H) [13,16] and the Heptosyltransferase II (HepII, pdb id = 1PSW) structures using the SALIGN program [17]. 2.5. Heptosyltransferase sequence and structure similarity analyses Initially, amino acid sequences of E. coli Heptosyltransferases I, II and III were aligned using ClustalW alignment program webserver with default settings. Based upon the structural alignments generated above, the ClustalW alignment was modified to ensure that the location of gaps and residues were consistent with their structural superposition, to aid in comparison of the three proteins. 3. Results and discussion 3.1. Soluble expression of HepIII Determination of conditions for the soluble expression of the E. coli HepIII was a surprisingly challenging task since both HepI

(A)

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and HepII from E. coli had previously been not only functionally characterized [5,18], but also structurally characterized (HepI pdb ids = 2GT1, 2H1H and 2H1F; HepII pdb id = 1PSW). In this work, we explored multiple vectors for the expression of HepIII (pET-15b, pET-21a, pET-17b and pNGFP-BC, corresponding to an N-terminal 6-Histitine fusion, a C-terminal 6-Histitine fusion, an untagged protein and an N-terminal green fluorescent protein (GFP) fusion, respectively). We also tested a variety of different bacterial cell lines (E. coli BL21 (DE3) cells – New England Biolabs, Ipswich, MA; E. coli BL21-AI cells – Life Technologies, Grand Island, NY; and E. coli Arctic Express cells) with growths ranging from 5 to 24 h at temperatures ranging from 10 to 37 °C. Only the N-GFP-HepIII fusion protein exhibited soluble expression when expressed in E. coli Arctic Express cells which were grown at 10 °C after induction with ITPG. As was previously observed by our group for Heptosyltransferase I, HepIII had high non-specific affinity to resins and membranes that were sugar based. As such, attempts to purify HepIII with a column containing traditional agarose based affinity column resin would lead to the protein being retained on the column under all conditions except treatment with 6 M urea. This protein was, however, able to be successfully purified by Histidine tag affinity chromatography using a Toyopearl AF Chelate column charged with Cobalt, which is a functionalized non-sugar hydrophilic resin (Fig. 2A). Building off our experience with HepI, we then selected Vivaspin concentrators because of their non-sugar polymer components for the concentration of HepIII. After affinity chromatography and buffer exchanging, HepIII could be obtained at high purity at concentrations of up to 5.2 mg/mL (Fig. 2B). Once we obtained pure N-GFP-HepIII fusion protein, multiple attempts were made to cleave off the GFP fusion; however, all efforts yielded stoichiometric precipitation of HepIII (the GFP protein stayed soluble). Based upon these results we proceeded to perform kinetic characterization of the fusion protein with the recognition that activity might be altered by the presence of the GFP protein. 3.2. Characterization of HepIII kinetic profiles On the basis of experiments with Heptosyltranferase I (HepI), we predicted that HepIII might well be able to use a variety of sugar acceptor (nucleophilic substrate) analogues due to the expectation that the hydrophobic acyl groups of the growing Lipid A would endogenously be intercalated into the lipid membrane. As such, we prepared fully acylated

(B)

75 50 37 25

Fig. 2. SDS page gel analysis of (A) the fractions produced as part of the purification of HepIII and (B) the final buffer exchanged and concentrated HepIII fraction. Untagged HepIII is 38.7kDa, and the N-GFP-HepIII fusion protein is 68.3kDa.

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Fig. 3. Steady-state kinetic profiles of the N-GFP-HepIII fusion protein with (A) PH2K2LA and (B) ODH2LA. Substrate concentrations above 25 lM were not used due to the high level of noise due to lipid induced light scattering.

using PH2K2LA or ODH2LA we were unable to obtain reliable rate data above 25 lM due to light scattering caused by micelle formation or other possible distortions due to the presence of the lipid tails, consistent with previous reports for the HepI substrate analogues [4]. Using this modified concentration range, we determined the kcat/Km for PH2K2LA to be 4.1  104 M1 s1 in the absence of detergent and 8.2  103 M1 s1 in the presence of 0.1% Triton X-100 (Table 1). This was surprising to us because most prior work on enzymes involved in LPS biosynthesis have been observed to have enhanced catalysis in the presence of detergents, either because of stabilization of the enzyme, or because of favorable interactions with the acylated substrates [2,8,19,20]. Alternatively, it is possible, however, that because HepIII acts on a substrate that is further removed from the membrane than other previously studied LPS biosynthetic enzymes, that it interacts less with the membrane and is therefore not stabilized by detergents which can help encapsulate the Lipid A analogue substrates and mimic a membrane surface. Removal of the O-linked acyl groups caused almost no change in the catalytic efficiency for HepIII when compared with the native acceptor substrate, with a kcat/Km for ODH2LA of 4.6  104 M1 s1 in the absence of detergent and 7.9  103 M1 s1 in the presence of 0.1% Triton X-100 (Table 1). Interestingly, a greater

Table 1 Steady-state kinetic parameters measured with PH2K2LA and analogous substrates either in the presence or absence of Triton X-100. Substrate

kcat (s1)

Km (lM)

kcat/Km (M1 s1)

PH2K2LA PH2K2LA + Triton X-100 ODH2LA ODH2LA + Triton X-100 FDH2LA

0.41 ± 0.03 0.049 ± 0.008 0.028 ± 0.001 0.019 ± 0.002 0.07 ± 0.02

10 ± 2 6±3 0.6 ± 0.1 2.4 ± 0.8 10 ± 5

4.1 ± 0.9  104 8.2 ± 4.3  103 4.6 ± 0.8  104 7.9 ± 2.8  103 6.7 ± 4.0  103

phospho-Hep2-Kdo2-Lipid A (PH2K2LA) as well as a partially and a fully deacylated analogue (ODH2LA and FDH2LA, respectively) using methods analogous to those for purification of the Lipid A substrate of HepI (Fig. 3) [8–10]. We then assessed the catalytic efficiency of each of these substrates with the N-GFP-HepIII fusion protein, in the presence of 100 lM ADP-Hep (Table 1 and Fig. 4). Additionally, we assessed the impact of 0.1% Triton X-100 detergent on the kinetics of the fully and partially acylated substrates (PH2K2LA and ODH2LA), since we had observed in HepI an enhancement in overall catalytic efficiency for acylated substrates in the presence of detergent [8]. While we had initially attempted to perform kinetic characterization over the concentration range of 200 nM to 100 lM, when

HO HO HO

OH OH O

OH

O HO O HO P O O OH HO HO

OH O

OH OH O

HO HO HO

Hep-II OH O

OH

O O

HO OH

O HO

O

O

HO

O

OH O

O

O O

O

10 12 O

O

O O

NH HO O O HO 10

10 phospho-Hep2-Kdo2 -LipidA (PH2K2LA)

O

NH O

10 O

OH O

O OH P O

10 14

Hep-II

OH

O HO OH

HO

O

O

O O

OH O

O O

NH HO HO

NH

HO

O

OH O

Hep-I O

O O HO

O

O O O P O HO O HO

OH

O HO O HO P O O

O

O OH P O

OH O

O O

O O P O HO O HO

O 10

O

OH OH O

Hep-I

O HO O

O O O P O HO OH O O

OH

O HO O HO P O O

Hep-I

HO HO HO

Hep-II

O

O

NH2 HO HO

O

O

NH2

HO

10 O-deacylated phospho-Hep2-Kdo2-LipidA (ODH2LA)

fully deacylated phospho-Hep2-Kdo2-LipidA (FDH2LA)

Fig. 4. Structure of the HepIII substrates used in the study (A) PH2K2LA, (B) ODH2LA and (C) FDH2LA.

O OH P O

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Fig. 5. Superposition of the nine HepIII structural models (model1 = tan; model2 = plum; model3 = gray; model4 = dodger blue; model5 = yellow; c2h1fB = light blue; d1pswA = spring green; c3tovB = coral; c4rapD = deep pink). The C-terminus is on top and the N-terminus is on bottom, with the models depicted looking (A) down the top Rossmann-like domain and (B) rotated 90°.

Fig. 6. Superposition of HepI (pdb id = 2H1H) and HepII (pdb id = 1PSW) with model1 (tan) and c3tovB (coral). The C-terminus is on top and the N-terminus is on bottom, with the models depicted looking (A) down the top Rossmann-like domain and (B) rotated 45° to accentuate the differences in the prediction for residues 295–320.

than 10-fold decrease in kcat for the O-deacylated substrate compared to the fully acylated version (corresponding to rates of 0.028 ± 0.001 and 0.41 ± 0.03 s1, respectively) was more than off-set by compensating decreases in Km. While this decrease in kcat observed for HepIII is more pronounced than in HepI, none-the-less the overall catalytic rate for the deacylated substrate is better when at least some of the acyl groups have been removed. Further, we determined the kinetic profile for FDH2LA, and observed that while kcat was reduced as compared to PH2K2LA, there was not a compensating increase in affinity for the fully deacylated substrate as observed in ODH2LA. We hypothesize that the negative impact of the newly revealed positive charges on the amines of FDH2LA could be counteracting any gains in affinity from removal of preexisting negative interactions between the charged surface of HepIII with the hydrophobic side chains of PH2K2LA, making ODH2LA a better substrate overall.

3.3. HepIII structure and sequence comparisons While efforts have been made to crystallize the N-GFP-HepIII fusion protein, diffraction quality crystals have been elusive. To aid the interpretation of our kinetic data, multiple structural models of HepIII were generated using the Phyre2 [14] and I-TASSER [15] servers (Fig. 5), both of which are top performing programs in the CASP (Critical Assessment of methods of protein Structure Prediction) structure prediction competitions [21,22]. All of the models generated maintained the general GT-B structural architecture, defined as two Rossmann-like domains connected by a typically alpha-helical linker. When comparing the nine models generated, the most variable region corresponds to amino acids 295–320, which is alternately defined as a loop, as one or more alpha helices or a pair of beta strands; these residues correspond to a short beta strand in HepI and a disordered loop in HepII.

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Fig. 7. Sequence alignment of HepI, HepII and HepIII. Residues that are known to be important for catalysis are highlighted in red. Residues that are strictly conserved are highlighted dark blue and that are similar are in light blue. The regions representing beta strands are indicated by a green bar, and those representing alpha helices are indicated by a purple bar. The linker region is indicated by a yellow bar.

Each of the structural models generated was then simultaneously aligned with both HepI and HepII (pdb ids = 2H1H and 1PSW, respectively). For two of the nine models (model1 generated by I-TASSER and model c3tovB generated by Phyre2), the root-mean square deviation was less than 2.0 Å to both crystal structures (model1 had an RMSD of 1.32 and 1.86 Å with HepI and HepII, while c3tovB had an RMSD 1.69 and 1.77 Å with HepI and HepII, respectively; Fig. 6). Both of these models nicely traced the backbone of both Rossmann-like domains and the linker, with the primary difference being the positioning of the alpha helix and loop corresponding to residues 295–320. Based upon the structural alignment of these models with the previously determined X-ray crystal structures, a protein sequence alignment was generated from the consensus alignment of these two models with HepI and HepII (Fig. 7). The N-terminal Asp that is important for catalysis and the C-terminal residues that are implicated in coordinating the ADP-Hep are conserved. Additionally, the relative residue conservation or similarity varies in each part, with the C-terminus having the most conservation (likely due to the preserved role of binding ADP-Hep), followed by the N-terminus, with the linker region being the most variable (both in residue identity and number of residues). Next, we examined the N-terminal domain in an attempt to use the structure and sequence information to predict the binding site of the sugar acceptor substrate. In particular, upon looking for positively charged regions that might stabilize the three phosphate residues or the two carboxylates in PH2K2LA, three clusters of Lys and Arg residues were identified (Fig. 8; the first composed of residues 54–72, second composed of residues 93–115, and a third composed of residue 131 in the N-terminus and residues 301–313 from the C-terminus which would be predicted to be proximal as HepIII undergoes an open-to-closed transition characteristic of the GT-B superfamily members [4,23,24]). Additionally, it was observed that there was a greater preponderance of negatively charged residues in the C-terminal domain, as opposed to the N-terminal domain

Fig. 8. Structural representation of model1 showing the Arg and Lys residues that are clustered together and could represent the regions where the negatively charged phosphate and carboxylate groups would localize when the sugar acceptor substrate binds to HepIII. The first cluster is composed of residues 54–72 (orange), the second is composed of residues 93–115 (black), and a third composed of residue 131 in the N-terminus and residues 301–313 from the C-terminus (magenta).

(the N-terminus has 7 Asp and 4 Glu residues, while the C-terminus has 13 and 8, respectively). The relative density of positively charged residues in the N-terminus and the negatively

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charged residues in the C-terminus, reinforces our hypothesis as to why the fully deacylated FDH2LA is not as tightly bound as ODH2LA which is only negatively charged or neutral in various positions. We hypothesize that the different overall charge states of the two Rossmann-like domains might help establish a dipole across the protein reinforcing the likelihood of the N-terminal domain being pointed toward the membrane to aid in the search for the membrane bound substrate. Further investigation of HepIII and other Lipid A derivatizing enzymes could reveal if the charge distribution pattern is conserved and if it is important for either the bimolecular association and binding of the sugar acceptor substrate. This work provides the first characterization of the Heptosyltransferase III from E. coli. The enzyme was shown to be kinetically competent with a variety of sugar acceptor substrates, with PH2K2LA and ODH2LA having the highest catalytic proficiency. These experiments inform questions about the mechanisms by which LPS biosynthesis and more specifically how Heptosyltransferase enzymes work. Studies further comparing the three Heptosyltransferases, all of which form different glycosidic bonds using similar enzyme architecture and the same sugar donor, should be performed in the future so that the chemical and mechanistic differences can be better understood. Acknowledgements We thank Chris Whitfield (University of Guelph) for the E. coli HepIII (WaaQ) deletion strain, CWG297, Eric Gouaux (Vollum Institute of the Oregon Health & Science University) for the pNGFP-BC plasmid, and Wesleyan University for financial support. Appendix A. Supplementary data ESI-MS data for the HepIII substrates are provided. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.04. 051. References [1] Petrovsky, N. and Aguilar, J.C. (2004) Vaccine adjuvants: current state and future trends. Immunol. Cell Biol. 82, 488–496. [2] Zhao, G., Wu, B., Li, L. and Wang, P. (2014) O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis. Appl. Microbiol. Biotechnol. 98, 4075–4081. [3] Chen, L. and Coleman, W.G. (1993) Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis. J. Bacteriol. 175, 2534–2540. [4] Czyzyk, D.J., Sawant, S.S., Ramirez-Mondragon, C.A., Hingorani, M.M. and Taylor, E.A. (2013) Escherichia coli heptosyltransferase I: investigation of protein dynamics of a GT-B structural enzyme. Biochemistry 52, 5158–5160.

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