Effect of Acceptor Chain Length and Hydrophobicity on Polymerization

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Effect of Acceptor Chain Length and Hydrophobicity on Polymerization Kinetics of the Neisseria meningitidis Group C Polysialyltransferase Shonoi Ming, Natalee Caro, Nicholas Lanz, Justine Vionnet, and Willie Vann Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01114 • Publication Date (Web): 02 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Biochemistry

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Effect of Acceptor Chain Length and Hydrophobicity on Polymerization Kinetics of the Neisseria meningitidis Group C Polysialyltransferase

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Running title: Polymerization kinetics of Neisseria polysialyltransferase

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Shonoi A. Ming, Natalee C. Caro, Nicholas Lanz, Justine Vionnet and Willie F. Vann*,

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Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Silver Spring, Maryland 20993

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*Corresponding author, email address: [email protected]

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Keywords: polysialyltransferase, polysialic acid, nucleotide monophosphate kinase

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ABSTRACT

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Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens

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Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in

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virulence make the polysialyltransferases (PST) that synthesize them targets for

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therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here,

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we have generated recombinant bovine nucleotide monophosphate kinase to facilitate

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steady state kinetic assays of the PST. We have characterized the N. meningitidis Group C

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(NmC) PST kinetically, using substrate analogs to describe the polymerization reaction. We

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observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased,

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indicating a tighter binding of longer oligomers. In addition, we observed a biphasic

12

relationship between kcat and chain length, which can be attributed to a switch in the

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mechanism of transfer of sialic acid from distributive to processive as the chain length

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increased above six sialic acid units. Substitution of donor substrate with the analog CMP-

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9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose

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that this decrease in kcat is cause by a destabilization of the transition state and/or an

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increase affinity of the product due to presence of the fluoro substituent. The acceptor’s

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hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with

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hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned

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over at a faster rate than the α-2, 9 polysialic acid substrates lacking the hydrophobic end.

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This finding suggests the presence of a secondary ligand binding site that tethers the

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acceptor substrate to the enzyme active site.

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Biochemistry

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INTRODUCTION

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Neisseria meningitidis is a Gram-negative human pathogen that was discovered to be the

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causative agent of meningococcal meningitis at least 200 years ago1. It is carried by 8-25%

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of the population in their pharynx, with adolescents being the main reservoir2. The

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symptoms of the disease mimic those of the flu; the only notable symptom that

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distinguishes it clinically is a stiff neck. This contributes to a delay in treatment, and even

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with treatment, the infection is lethal in approximately 1 in 10 cases.

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As is the case for other human pathogens, N. meningitidis can be encapsulated with an

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extracellular polysaccharide structure. Due to its surface exposure, the bacteria’s capsule is

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first portion of the organism that encounters the host immune system, serving as a physical

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barrier to host defense and in some cases to disguise the bacteria from the host immune

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system3.

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Antibodies produced against N. meningitidis polysaccharide capsule can be protective4.

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Because protective antibodies can be generated against the abundant polysaccharide on

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the bacterial surface, the capsule is a common target for vaccine development. Due to the

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critical role of the capsule structure in pathogenesis, the enzymes involved in its

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biosynthesis serve as excellent targets for therapeutics.

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At least twelve serogroups have been defined based on their capsular polysaccharide

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structures5. Of the twelve serogroups, six of these (A, B, C, X, Y and W-135) are responsible


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for almost cases of invasive disease. Serogroups B and C are responsible for most cases of

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meningococcal meningitis in Canada, Europe and the United States6.

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The gene cluster responsible for the synthesis of the polysaccharide capsule has been

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identified and characterized7, 8. The capsular polysaccharide synthesis and cell surface

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translocation genes are clustered on a single chromosomal locus termed cps. The locus is

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divided into six regions (D, A, C, E, D’ and B). Regions A, C, and B have been associated with

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capsule synthesis, transport, and translocation functions respectively. However, the genes

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in region A are specific for serogroup polysaccharide structure and are therefore diverse8.

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The polysialic acid capsules of serogroups B and C, as well as those of E. coli K1 and K92,

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are synthesized by linkage-specific polysialyltransferases of the CAZy glycosyltransferase

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family GT-389. The NmC and NmB polysialyltransferases share 75% sequence similarity

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and 64% identity. NmC PST synthesizes the α2,9 homopolymer, while NmB PST and the E.

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coli K1 polysialyltransferases synthesize the α2,8 homopolymer. However, there is only

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30% sequence identity between the E. coli K1 and NmB polysialyltransferases, and yet E.

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coli K1 and NmB synthesize identical polymers10. Previous studies conducted in our

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laboratory and others have found that the amino acid sequence at residues 1-107 of the

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polysialyltransferases was enough to dictate the orientation of the linkage of the product11,

18

12.

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We have characterized the NmC polysialyltransferase reaction using steady state kinetics

20

to gain further insight on the reaction mechanism of bacterial polysialyltransferases. We

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found a shift in the mechanism of acceptor elongation from distributive to processive with

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increased acceptor chain length. In addition, we observed an increase in kinetic activity


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Biochemistry

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towards acceptor substrates that contain a hydrophobic moiety. This finding along with the

2

structure of the natural acceptor proposed by Willis et. al13, has led us to hypothesize the

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presence of a secondary lipid binding site within the active site of the

4

polysialyltransferases.

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MATERIALS AND METHODS

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Materials

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5-SFX (6-(Fluorescein-5-Carboxamido) Hexanoic Acid, Succinimidyl Ester), single isomer

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(FCHASE) was purchased from Thermo Fisher Scientific. 4-amino-lactopyranoside was

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purchased from Carbosynth (Compton, Berkshire, UK). Cytidine-5'-monophospho-N-

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acetylneuraminic acid (CMP sialic acid) disodium salt and α2, 8 sialic acid hexamer (DP6)

9

disodium salt was purchased from Nacali Tesque (San Diego, CA). 9-F-sialic acid was

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purchased from TCI chemicals (Shanghai, China). Inorganic pyrophosphatase and bovine

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alkaline phosphatase were purchased from Sigma. The ganglioside GD3 was purchased

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from Avanti polar lipids Neisseria meningitidis CMP sialic acid synthetase (CSS) was

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expressed and purified as previously described by Karwaski et al. 14 Unless noted

14

otherwise, all other reagents were purchased from commercial sources and used without

15

further purification.

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Bacterial strains

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The bacterial strains and plasmid pWV243 used in this study have been described

18

previously by Peterson et al.12 A plasmid for expression of bovine liver nucleotide

19

monophosphate kinase was custom synthesized by Geneart based on a codon optimization

20

of the published sequence of NMPK (UniProt Accession: Q2KIW9).15, 16


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Biochemistry

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Expression and purification of NmC PST

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E. coli BL21(DE3) cells were transformed with the pWV243 plasmid containing the NmC

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PST gene. LB broth (1 L) supplemented with 100 μg/mL ampicillin was inoculated with 10

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mL of an overnight (o/n) inoculum. The culture was incubated at 37 °C, while shaking at

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170 rpm until an optical density at 600 nm (OD600) of 0.8-1.0 was achieved. Subsequently,

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the culture was induced with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and

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incubated further at 20 °C for 18-20 h. The cells were harvested by centrifugation at 10,000

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× g for 15 min at 4 °C, followed by resuspension in 100 mM Tris pH 8.0. The cell suspension

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was lysed by passage through a French pressure cell and centrifuged for 20 min at 10,000 ×

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g at 4 °C. Membranes were separated from the cytosolic fraction by ultracentrifugation for

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1 h at 165,000 × g at 4 °C. The supernatant was loaded onto a MBPTrap™ HP (GE

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Healthcare) column, equilibrated with 20 mM Tris, 0.2 M NaCl, 1 mM EDTA, pH 8.0. The

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column was subsequently washed with the afore mentioned equilibration buffer and NmC

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PST was eluted with 10 mM maltose in the same buffer. The polysialyltransferase activity

15

of the eluted fractions was determined using a paper chromatography assay, previously

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described by McGowen et al17. The purified NmC PST solution was adjusted to 10%

17

glycerol, aliquoted, and stored at -80 °C.

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Preparation of α2,9 linked sialic acid oligosaccharides (DP2-DP-11)

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N. meningitidis C (NmC) polysaccharide was isolated and purified from NmC strain C11 as

20

previously described by Vann and Freese18. A 2.5 mg/mL solution of the NmC

21

polysaccharide was hydrolyzed for 20 min in 0.1 M acetic acid at 70 °C. The hydrolysis

22

reaction was halted by flash freezing the reaction mixture with dry ice/ethanol. The


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reaction mixture was lyophilized and redissolved in 20 mM Tris, pH 7.4. The resulting

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oligosaccharides were purified by HPLC on a 1 mL Mono Q™ GL ion-exchange column with

3

a linear gradient of 10-60% 1 M NaCl in 20 mM Tris, pH 7.4. The purified oligosaccharides

4

were desalted on a Bio-Gel®P2 column (100 cm × 0.75 cm).

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Preparation of alternating α2,8/2,9 linked sialic acid hexamer

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The K92 alternating α2,8/2,9 polysaccharide was isolated and purified from E. coli Bos 12

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as described by Vann and Freese19. The α2,8/2,9 sialic acid hexamer was prepared by

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digesting the α2,8/2,9 polysaccharide with the K1-5 endoneuraminidase as previously

9

described by McGowen et al17. The K1-5 endoneuraminidase is specific for the α2,8 sialic

10

acid linkage thereby producing an α2,8/2,9 sialic acid hexamer which terminates in the

11

α2,9 linkage.

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Synthesis of CMP -9-F-sialic acid

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CMP-9-F-sialic acid was synthesized by a modification of the method described by Morley

14

and Withers20. One equivalent of 9-F-sialic acid and 1.06 equivalents of CTP were combined

15

in buffer containing 100 mM bicine, 200 mM MgCl2, pH 9.0, resulting in final concentrations

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of 30 mM 9-F sialic acid and 32 mM CTP, respectively. In addition, the reaction mixture

17

contained 10 U inorganic pyrophosphatase and 1.8 U CMP sialic acid synthetase (CSS). The

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reaction was allowed to proceed for 30 min at 37 °C. The reaction progress was monitored

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by TLC with an ethyl acetate/methanol/water/ammonium hydroxide (4:3:2:1) solvent

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system. The TLC plate was sprayed with a 1:9 sulfuric acid/ethanol solution. The TLC plate

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was subsequently heated to greater than 150°C to visualize the 9-F-sialic acid spot. Upon

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consumption of all the 9-F-sialic acid, the reaction was halted by freezing at -80 °C. The


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Biochemistry

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reaction mixture was thawed and centrifuged to remove precipitate. The supernatant was

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transferred to a clean reaction vial, and 100 U alkaline phosphatase were added and

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incubated for 10 min at 37 °C to destroy residual CMP. Crude CMP-9-F-sialic acid was

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purified on a Bio-Gel®P2 column (25 cm × 0.75 cm) equilibrated in water. Fractions

5

containing CMP-9-F-sialic acid were identified by TLC, pooled, and lyophilized, yielding the

6

final product with >80% purity.

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Synthesis of GD3-FCHASE

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GD3-FCHASE was synthesized enzymatically by combining FCHASE-aminophenyl lactoside

9

and CMP-sialic acid in the presence of the enzyme CSTII and purified by TLC as described

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by Mosley et al21. The FCHASE-aminophenyl lactoside was prepared as described by

11

Wakarchuk et al22.

12

Determination of NmC PST kinetic parameters for the acceptor substrate

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Initial velocities for the transfer of sialic acid from a donor substrate (CMP-sialic acid or

14

CMP-9-F-sialic acid) to an acceptor substrate (GD3, α2,9-, α2,8- or α2,8/2,9- linked sialic

15

acid oligosaccharide) was determined using either a a) continuous multistep coupled assay

16

or a b) discontinuous HPLC assay. Values for kcat and Km were obtained from the non-linear

17

least squares fit to the Michaelis-Menten equation.

18

a) The continuous coupled enzyme assays were performed as described previously, with

19

minor modifications.10, 23, 24 The initial reaction rates were monitored on a dual beam

20

Jasco V650 UV-Vis spectrophotometer equipped with a Peltier temperature system. The

21

enzymatic assays were conducted at 25 °C in buffer containing 50 mM Tris, 25 mM


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MgCl2, pH 8.0. The sample and reference cells contained 0.2 mM phosphoenolpyruvate

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(PEP), 0.3 mM NADH, 2 mM ATP, 24 U pyruvate kinase/lactate dehydrogenase

3

(Aldrich), 0.08 U of the nucleotide monophosphate kinase, 1 mM CMP sialic acid, and 2-

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267 M of one of the following acceptor substrates: GD3, α2,9-, α2,8- or α2,8/2,9- sialic

5

acid oligosaccharide. The reaction was initiated by the addition of NmC PST to a final

6

concentration of 5 nM in the sample cuvette.

7

b) The discontinuous enzyme assays were conducted at room temperature in 50 mM Tris,

8

25 mM MgCl2, pH 8.0. The reactions contained 1.0 mM CMP sialic acid or 9-F-CMP sialic

9

acid and the varied amounts of the fluorescently labeled GD3-FCHASE acceptor

10

substrate from 5-200 µM. Reactions were initiated by the addition of 5 nM NmC PST

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and allowed to proceed for 5 min (≤10% turnover). The reaction mixtures were

12

quenched by the adjusting to 25% ethanol. The quenched reactions were analyzed by

13

anion-exchange chromatography on a DNAPac™ PA100 oligonucleotide column with a

14

2-35% linear gradient of 1M NaNO3 over a period of 120 min.

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c) We confirmed that the chain is terminated in the discontinuous assay as follows. A

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mixture containing 0.3 M GD3-FCHASE, 1 mM CMP-9F-sialic acid, and 400 nM NmC

17

PST, in the above buffer was incubated 25 °C for 10 min, then quenched with 25%

18

ethanol. The reaction mixture was analyzed by anion-exchange chromatography as

19

above. The oligosialic acid product eluting at 31 min was collected, and purified by

20

applying to a C-18 Sep Pak. After washing the C18 with several column volumes of

21

water to remove salts, the product was eluted with 50% acetonitrile dried, and

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dissolved in 100 L water, of which 50 L was retained as a reference. The remaining

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oligosaccharide product was dried and tested for chain termination by dissolving in a


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Biochemistry

1

mixture containing 0.3 mM GD3-FCHASE, 1 mM CMP-sialic acid, and 400 nM NmC PST,

2

in 50 mM Tris, 25 mM MgCl2, pH 8.0 and incubated at 25 °C for 10 min. The reaction

3

was quenched with ethanol and analyzed by anion-exchange chromatography as

4

described above.

5

Determination of NmC PST kinetic parameters for the donor substrate

6

Initial velocities for transfer of sialic acid from CMP-sialic acid (24 µM to 1 mM) to GD3

7

(200 µM) catalyzed by NmC PST was determined using the continuous coupled enzyme

8

assay described above. The reactions were initiated with the addition of 35nM NmC PST.

9

Values for kcat and Km were obtained from the non-linear least squares fit to the Michaelis-

10

Menten equation.

11

Alternate Source for Nucleotide Monophosphate Kinase

12

During the course of these studies bovine liver nucleotide monophosphate kinase was

13

discontinued from commercial sources. Consequently, we developed a method of preparing

14

a highly active preparation of the bovine liver enzyme using recombinant technology in E.

15

coli. The gene encoding the bovine NMPK was codon-optimized for E. coli and synthesized

16

by GeneArt (Life Technologies, Carlsbad, CA) in the pET151-D-TOPO expression vector. In

17

this vector, the gene for NMPK is translationally fused to an N-terminal hexahistidine tag

18

with a 26-amino acid linker between the tag and the initiating methionine of NMPK. The

19

details of method of preparation and comparison of this enzyme preparation with

20

commercial enzyme is described in supplemental materials. The recombinantly purified

21

enzyme was found to be approximately 280-fold more active than the much less pure

22

commercial enzyme preparation from bovine liver (Figures S1 and S2).


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RESULTS AND DISCUSSION

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NmC PST Acceptor Substrate Selectivity. In previous studies conducted by our laboratory,

3

we demonstrated that NmC and E. coli K92 polysialyltransferases use a variety of polysialic

4

acids as acceptors12, 17. These studies were conducted using a discontinuous assay with

5

long assay times under non-steady state conditions. To explore the selectivity of NmC PST

6

with regards to the linkage of its acceptor substrate in a more quantitative fashion, we

7

conducted assays with hexameric sialic acid containing either an α2,8, α2,9, or alternating

8

α2,8/2,9 linkage under steady state conditions using our continuous assay described above

9

As expected, the hexamer containing the natural α2,9 linkage found in NmC was a substrate

10

for the NmC PST. In addition, both the α2,8-linked and α2,8/2,9-linked sialic hexamers

11

were also substrates for the enzyme. These findings are consistent with previous studies, in

12

which the polysialyltransferase enzymes were shown to contain a promiscuous acceptor

13

binding site, which allowed for elongation of α2,8-, α2,9-, or α2,8/2,9-linked polysialic acid

14

chains irrespective of the linkage the enzyme synthesizes11, 12. The enzyme adds α2,9

15

linkages regardless of the structure of the priming acceptor. In addition to generating the

16

most product with the α2,8-linked substrate, a kinetic analysis of the reaction revealed that

17

the NmC PST uses the α2,8-linked substrate with the highest efficiency. A comparison of

18

kcat/Km, a measure of catalytic efficiency, of NmC PST towards the varied acceptor substrate

19

linkages revealed that the enzyme has a strong preference for the α2,8-linked substrate

20

(Figure 1 and Table 1). The reaction of the NmC PST with the natural α2,9-linked substrate

21

displayed a Km of 489 ± 109 µM and kcat/Km of 3.2 µM-1min-1. As might be expected, the

22

α2,8/α2,9-linked substrate produced a significantly lower kcat/Km of 0.9 µM-1min-1.

23

However, it should be noted this lower value in kcat/Km was due to a decreased value in kcat.


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Biochemistry

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Conversely, the α2,8-linked substrate yielded a much higher kcat/Km of 10.7 µM-1min-1. This

2

increase in catalytic efficiency was due entirely to a 4-fold decrease in Km (138 ± 32), since

3

the kcat values for the α2,8 and α2,9 hexamers were essentially equivalent (Table 1). It is

4

well established that bacterial polysialyltransferases are incapable of initiating polysialic

5

acid synthesis, and an acceptor—at least a sialic acid dimer—is required for catalysis12, 17, 25,

6

26.

7

pathway which synthesizes this initiating acceptor molecule produces a molecule that is

8

linked in an α2,8 fashion. Further studies are required to confirm this hypothesis.

It is tempting, in light of these findings, to imagine that the enzyme on the biosynthetic

9 10 11

Figure 1. Comparison of kcat/Km of NmC PST for the acceptor substrates α2,8-, α2,9-, and α2,8/2,9-linked hexamer sialic acid hexamers.

12 13 14


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Table 1: Comparison of NmC PST kinetic parameters for the acceptor substrate using hexamer sialic acid containing an α2,8, α2,9 or alternating α2,8/2,9 sialic acid linkage. Kinetic parameters are determined at a constant donor concentration of 1mM CMP sialic acid with varying acceptor concentration from 2-267 µM. Hexamer linkage

Km (µM)

kcat(min-1)

kcat/Km (µM-1min-1)

α2,8

138 ± 32

1475 ± 182

10.7 ± 2.7

α2,9

489 ± 109

1586 ± 236

3.2 ± 0.9

α2,8/2,9

495± 101

460 ± 45

0.9 ± 0.2

5 6

Effect of acceptor chain length on kinetics of NmC PST reaction. Though it is well

7

established that bacterial polysialyltransferases are incapable of initiating polysialic acid

8

synthesis, the mechanism by which polymerization occurs and the length of the

9

oligosaccharide required for optimal activity is still unknown. To further characterize the

10

effects of oligosaccharide chain length, we determined the kinetic parameters for NmC PST

11

with the acceptor substrate using NmC α2,9-sialic acid oligosaccharides ranging in length

12

from 2-11 sialic acid residues.

13

We observed an increase in kcat/Km as the oligosaccharide increased in length from 2 to 4

14

sialic residues from approximately 0.1 µM-1 min-1 to 2.8 µM-1min-1. However,

15

oligosaccharides containing greater than 4 sialic residues showed no further increase

16

kcat/Km (Figure 2A). This indicates that no further enhancement in enzymatic activity is

17

gained by increasing the length of the oligosaccharide to more than 4 sialic acid residues.


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Biochemistry

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2 3 4 5

Figure 2. Comparison of NmC PST kinetic parameters for NmC α2,9-sialic acid oligosaccharides ranging in length from 2-11 sialic acid residues. (A) Comparison of kcat/Km values; (B) Comparison of Km values; (C)Comparison of kcat values.

6

On the other hand, Km decreased with increased chain length (Figure 2B), suggesting

7

tighter binding with each additional sialic residue. The catalytic turnover number, kcat,

8

displayed a biphasic pattern in which a gradual increase in kcat was observed as the

9

oligosaccharide chain length increased from 2 to 4 sialic acid residues. No change in kcat

10

was observed for oligomers containing between 4 and 6 sialic acid residues; however, kcat

11

decreased with each subsequent addition of sialic acid residues thereafter (Figure 2C). We

12

attribute this reduction in kcat to an increased affinity for the product, i.e. this heightened

13

binding of the product results in slow release from the enzyme active site and therefore a


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lower turnover number. The data suggest that the rate determining step of the enzymatic

2

reaction shifts from the conversion of the enzyme substrate complex (ES) to the enzyme-

3

product complex (EP) (Scheme 1) to the so-called release of the product (P) from the

4

enzyme product complex (EP). In the case of a processive mechanism (Scheme 2), the term

5

‘product release’ does not refer to the product leaving the enzyme, but rather the

6

repositioning of the product to shift the terminal sialic acid into the acceptor site within the

7

enzyme active site (ES′). This allows for another sugar-nucleotide molecule to bind in the

8

donor site for a subsequent round of turnover.

9

Scheme 1: Distributive Reaction Mechanism

ES

kcat

EP

10

E+S

11

Scheme 2: Processive Reaction Mechanism

E+P

12 13

We hypothesize that with oligomers ranging in length from 2 to 6 sialic acid residues the

14

NmC PST catalyzes polymerization in a distributive manner, where the product is released

15

after each transfer of sialic acid. In contrast, in this model, oligomers with greater than 6

16

sialic acid residues allow the enzymatic reaction to proceed in a processive manner with

17

slower product release, where multiple sialic acid transfers occur prior to the release of the

18

product.

19

Effect of acceptor substrate hydrophobicity on enzymatic activity. Although bacteria do

20

not naturally synthesize them, it has been reported that glycosphingolipids can be suitable


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Biochemistry

1

acceptor substrates for bacterial polysialyltransferases12, 17, 25. Gangliosides that have

2

disialic acid residues such as GD3 and GT1a are the best substrate analogs for these

3

enzymes. We have investigated the kinetics of GD3 as an acceptor substrate for NmC PST.

4

Our results showed GD3 to be a better substrate than the best oligosaccharide acceptor, α

5

2,9 sialic acid undecamer used in the experiments described above. A kcat of 645 ± 25 min-1

6

was determined for GD3. This value is 3-fold greater than that determined for the α 2,9

7

sialic acid undecamer acceptor (kcat = 203 ± 11 min-1). In addition, a kcat/Km of 58 ± 1 µM-

8

1min-1

9

undecamer acceptor. This result indicates that even though GD3 contains only a sialic acid

was determined for GD3 which is 22-fold higher than that observed with the

10

dimer in its structure, the elongation of the ganglioside acceptor is faster and more

11

efficient. This finding, though somewhat surprising, agrees with the previously published

12

reports that found that short oligomers without a lipid attached were poorer substrates

13

than oligomers with a lipid attached. These short oligomers were reported by Cho and

14

Troy to be extended by the E. coli K1 polysialyltransferase by only a few sialic residues,

15

while the lipid-attached oligomers readily formed long polymers25, 27. Additionally, the

16

activity of the NmC PST was increased 10 fold when GD3 was used as acceptor substrate,

17

compared with the non lipidated K92 hexamer oligomer containing an α2,9 disialic acid

18

linkage at its non-reducing end12. We attribute the increased activity with GD3 to a

19

hydrophobic substrate interaction of GD3 with the NmC PST’s acceptor binding site. The

20

ceramide moiety of GD3 may mimic the proposed phospholipid anchor28 of the NmC

21

polysaccharide capsule to improve the binding of the acceptor and in turn increase the

22

catalytic efficiency of the enzyme. However, it is possible that GD3 may form micelles

23

under our reaction conditions and hence influence the outcome of the assay. Nevertheless,


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

the formation of micelles would lead to a decrease in the effective concentration of GD3

2

present for the enzymatic reaction. Such a reduction in the effective concentration of GD3

3

would cause the activity observed with NmC PST to be underestimated. Therefore, we don’t

4

believe the formation of micelles would have positive impact on NmC activity as we have

5

observed, but on the contrary, a decrease in activity would result.

6

An analysis of the reaction of the NmC PST reaction with a fluorescently-tagged GD3-

7

FCHASE acceptor by HPLC visually depicts the shift in the mode of polymerization. In a

8

previous study conducted by Peterson et al. a time course experiment where GD3-FCHASE

9

was incubated with NmC PST for 2 mins, 5 mins and 15 mins was performed followed by

10

HPLC analysis of the reaction mixture.12 The HPLC traces from this experiment show the

11

conversion of the starting material (GD3-FCHASE) to small oligosaccharides that can be

12

separated into individual peaks for each additional sialic acid appended. As the reaction

13

time is increased, significant quantities of large polymers that are greater than 100 sialic

14

acid residues in length can also be observed in the trace as a high molecular weight peak

15

near 100 min. Notably, polymers up to 17 sialic residues in length as well as polymers >100

16

sialic acid residues were observed; however, polymers of intermediate length are present

17

in much lower amounts. This finding suggests that oligomers greater than 17 sialic acid

18

residues are elongated with multiple additions sialic acid with infrequent release from the

19

active site leading to formation of polymers greater than 100 sialic acid residues.

20

Therefore, we believe the findings from the Peterson et al. work support the proposed shift

21

in the mode of polymerization catalyzed by NmC PST from distributive to processive with

22

increased chain length, suggested above by the change in Km and kcat with acceptor chain

23

length. Interestingly, for E. coli K1 polysialyltransferase, Cho and Troy25 reported observing


Page 18 of 28

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Biochemistry

1

a few additions of sialic acid when short non-lipidated oligosaccharides were used as

2

acceptor substrates. In contrast, they observed long polymers with oligosaccharides that

3

contained a lipid moiety. These findings are similar to those observed by May et al. where

4

the presence of a lipid moiety on the acceptor substrate for the glycosyltransferase

5

galactofuranosyltransferase (Glft2) enhanced the enzymatic processivity and suggested

6

that the lipid serves as a tether to the enzyme active site29. They propose that the presence

7

of a secondary lipid binding site where lipid binding extends the enzyme-acceptor

8

substrate complex and facilitates a processive mechanism to produce longer polymers. The

9

phospholipid anchor of GD3 may similarly bind to a secondary lipid binding site in the NmC

10

PST and serve as a tether allowing for elongation in a processive manner. Though

11

additional studies are required to definitively prove the presence of a secondary lipid

12

binding site, we believe based on our results that the lipid moiety enhances the shift in the

13

mechanism of polymerization from distributive to processive that we have associated with

14

acceptor chain length.

15

NmC PST donor substrate kinetics. The kinetics of the other substrate for the α2,9

16

polysialyltransferase, the donor or CMP-sialic acid, also remain incompletely understood.

17

NmC PST belong to CAZy glycosyl transferase family GT-38. Other members of this family

18

include NmB PST as well as the E. coli K1 and K92 PSTs. The NmC and NmB

19

polysialyltransferases share 75% sequence similarity and 64% identity. It is postulated

20

that the findings from the study of one of these enzymes can be extended to the other

21

members of the family. All four enzymes utilize the donor substrate CMP-sialic acid.

22

However, early studies of other bacterial polysialyltransferases reported Km values for the

23

donor substrate CMP-sialic acid ranging from low µM to mid mM 28, 30, 31. A more recent


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

study conducted by Freiberger et al. obtained an apparent Km of 420 µM for CMP-sialic acid

2

for the α2, 8 polysialyltransferase from NmB 10. This is comparable to the Km value of 232 ±

3

21 µM we obtained using the α2,9 polysialyltransferase. The differences in Km values

4

between the earlier studies and those conducted more recently may be due to enzyme

5

preparation and varying assay conditions. The early studies were conducted using

6

membrane bound enzyme and more recent studies employed soluble recombinant enzyme.

7

In addition, early studies used a stopped radiolabeled assay, whereas this work and the

8

recent study conducted by Freiberger et al. were performed using a multistep coupled

9

continuous UV/Vis assay. The advantage of the continuous assay over the stopped

10

radiolabeled assay is that it allows the measurement of initial velocities. The previous

11

studies conducted using the stopped radiolabeled assay was halted at 60 mins, irrespective

12

of the amount the 14C CMP sialic acid was turned over. Therefore, the kinetic parameters

13

obtained using this method are less accurate.

14

Effect of a fluoro substitution at the C9 position of CMP-sialic acid. Kinetic analyses of

15

the NmC PST are made more complex by the nature of the reaction it catalyzes. The

16

product formed during one round of catalysis becomes a substrate for subsequent rounds.

17

As described above, the newly formed product likely has altered kinetic properties with

18

regard to Km and kcat, further complicating the analysis. This makes it challenging to

19

elucidate the effect of the addition of a single sialic acid transfer with the natural

20

substrates. Therefore, we used the substrate analog, CMP-9-F-sialic acid, to develop a

21

single-transfer assay, since this donor substrate allows for the addition of a single sialic

22

residue but prevents subsequent additions to the acceptor substrate by blocking the non-

23

reducing terminus. The transfer of the 9-F sialic acid from the donor substrate to the


Page 20 of 28

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Biochemistry

1

acceptor substrate forms an elongated product that is unable to accept further sialic acid

2

additions, since the 9-hydroxyl nucleophile is replaced with a fluoro substituent (Scheme

3

3).

4

Scheme 3

5

6 7 8

We assessed the effect of the 9-fluoro substitution on the enzymatic activity of the NmC

9

PST by the discontinuous HPLC assay described above. The NmC PST uses the 9-F-CMP-

10

sialic acid donor as a substrate to yield a product that has been elongated by a single

11

residue (Figure 3). Isolation of the elongated product and incubation with fresh enzyme

12

and CMP-sialic acid should no further elongation confirming the termination of chain

13

growth with a single transfer (Figure 3C), The steady state results obtained in this single-

14

transfer assay indicate that the Km for the acceptor was not significantly affected when the

15

ceramide moiety was replaced with the fluorescent FCHASE tag (Figure 4, Table 2). This

16

suggests that GD3-FCHASE binds in a similar manner to GD3 at the NmC PST’s acceptor

17

binding site. This also indicates that the acceptor binding pocket is both hydrophobic and

18

large enough to accommodate large acceptor substrate modifications. However, the kcat for

19

CMP-9-F-sialic acid was reduced by more than 5-fold with respect to CMP-sialic acid, and a


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

corresponding 10-fold decrease in kcat/Km was also observed. We propose that this change

2

in kcat is caused by a destabilization of the transition state and/or an increase in the affinity

3

for the product upon substitution of the 9-hydroxyl with the 9-fluoro substituent. An

4

increase in affinity of NmC PST for the 9-fluoro substituted product would lead to a delayed

5

release of the product from the enzyme active site. Thereby, the newly formed product now

6

serves as an inhibitor. In this scenario the slow release of the product could explain the

7

decrease in kcat observed with CMP-9-F-sialic acid.

8


Page 22 of 28

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1 2 3 4

Biochemistry

Figure 3. HPLC trace GD3-FCHASE chain termination reaction. A. GD3-FCHASE (control), B. NmC PST-catalyzed reaction mixture using CMP-9-F-sialic acid and GD3-FCHASE, C. NmC PST catalyzed reaction mixture using CMP-sialic acid and isolated product shown in panel B.

5

6 7 8 9

Figure 4. A. Structure of FCHASE moiety of GD3-FCHASE, B. Structure of ceramide moiety of GD3

10 11 12 13 14

Table 2: Comparison of kinetic parameters for unlabeled GD3 vs fluorescently tagged GD3FCHASE. Kinetic parameters are determined at a constant donor concentration of 1mM CMP sialic acid or CMP-9-F-sialic acid with varying acceptor (GD3 or GD3-FCHASE) concentration from 2-267 µM.

15

GD3 + CMP-Sia

GD3-FCHASE + 9-F-CMP-Sia

Km (µM)

11 ± 2

21 ± 4

18

kcat(min-1)

645 ± 25

118 ± 11

19

kcat/Km (µM-1min-1)

59 ± 2

6±1

16 17

20 21

Summary

22

In this study, we show that the NmC PST, despite synthesizing an α2,9 sialic acid linked

23

polysaccharide, has a strong preference towards α2,8 sialic acid as the acceptor substrate.

24

We speculate that this unexpected increase in affinity toward the α2,8 linkage may be


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

attributed to the linkage contained in the polymerization initiating molecule. We also

2

observed a shift in the mode of catalysis of acceptor elongation from distributive to

3

processive with increased acceptor chain length. Furthermore, the activity of the enzyme is

4

significantly improved when the GD3 ganglioside is used as the acceptor molecule,

5

suggesting the presence of a secondary lipid binding site within the active site of the NmC

6

PST. We have determined the kinetic parameters for NmC PST with its donor substrate

7

CMP-sialic acid and found our results to be in close agreement with those obtained for the

8

NmB polysialyltransferase. In addition, we employed the CMP-9-F-sialic acid donor

9

analogue to study the NmC PST reaction under single transfer conditions.

10 11

Associated Content

12 13

Supporting information: Detailed description of the expression, purification and characterization of recombinant Nucleotide Monophosphate Kinase (NMPK).


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Biochemistry

References [1] Stephens, D. S. (2009) Biology and pathogenesis of the evolutionarily successful, obligate human bacterium Neisseria meningitidis, Vaccine 27 Suppl 2, B71-77. [2] Rosenstein, N. E., Perkins, B. A., Stephens, D. S., Popovic, T., and Hughes, J. M. (2001) Meningococcal Disease, N. Engl. J. Med. 344, 1378-1388. [3] Troy, F. A., 2nd. (1992) Polysialylation: from bacteria to brains, Glycobiology 2, 5-23. [4] Goldschneider, I., Gotschlich, E. C., and Artenstein, M. S. (1969) Human immunity to the meningococcus. I. The role of humoral antibodies, J. Exp. Med. 129, 1307-1326. [5] Branham, S. E. (1953) Serological relationships among meningococci, Bacteriol. Rev. 17, 175-188. [6] Oviedo-Orta, E., Ahmed, S., Rappuoli, R., and Black, S. (2015) Prevention and control of meningococcal outbreaks: The emerging role of serogroup B meningococcal vaccines, Vaccine 33, 3628-3635. [7] Swartley, J. S., Ahn, J. H., Liu, L. J., Kahler, C. M., and Stephens, D. S. (1996) Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region, J. Bacteriol. 178, 4052-4059. [8] Harrison, O. B., Claus, H., Jiang, Y., Bennett, J. S., Bratcher, H. B., Jolley, K. A., Corton, C., Care, R., Poolman, J. T., Zollinger, W. D., Frasch, C. E., Stephens, D. S., Feavers, I., Frosch, M., Parkhill, J., Vogel, U., Quail, M. A., Bentley, S. D., and Maiden, M. C. (2013) Description and nomenclature of Neisseria meningitidis capsule locus, Emerging infectious diseases 19, 566-573. [9] Coutinho, P. M., Deleury, E., Davies, G. J., and Henrissat, B. (2003) An evolving hierarchical family classification of glycosyltransferases, J. Mol. Biol. 328, 307-317. [10] Freiberger, F., Claus, H., Günzel, A., Oltmann-Norden, I., Vionnet, J., Mühlenhoff, M., Vogel, U., Vann, W. F., Gerardy-Schahn, R., and Stummeyer, K. (2007) Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases, Mol. Microbiol. 65, 1258-1275. [11] Steenbergen, S. M., and Vimr, E. R. (2003) Functional relationships of the sialyltransferases Involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C, J. Biol. Chem. 278, 15349-15359. [12] Peterson, D. C., Arakere, G., Vionnet, J., McCarthy, P. C., and Vann, W. F. (2011) Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase, J. Bacteriol. 193, 1576-1582. [13] Willis, L. M., and Whitfield, C. (2013) KpsC and KpsS are retaining 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules, Proc Natl Acad Sci U S A 110, 20753-20758. [14] Karwaski, M. F., Wakarchuk, W. W., and Gilbert, M. (2002) High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli, Protein Expression Purif. 25, 237-240. [15] The UniProt Consortium. (2017) UniProt: the universal protein knowledgebase, Nucleic Acids Res. 45, D158-D169. [16] Zimin, A. V., Delcher, A. L., Florea, L., Kelley, D. R., Schatz, M. C., Puiu, D., Hanrahan, F., Pertea, G., Van Tassell, C. P., Sonstegard, T. S., Marçais, G., Roberts, M., Subramanian, P., Yorke, J. A., and Salzberg, S. L. (2009) A whole-genome assembly of the domestic cow, Bos taurus, Genome Biol. 10, R42. [17] McGowen, M. M., Vionnet, J., and Vann, W. F. (2001) Elongation of alternating α2,8/2,9 polysialic acid by the Escherichia coli K92 polysialyltransferase, Glycobiology 11, 613-620.


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[18] Vann, W. F., Daines, D. A., Murkin, A. S., Tanner, M. E., Chaffin, D. O., Rubens, C. E., Vionnet, J., and Silver, R. P. (2004) The NeuC protein of Escherichia coli K1 is a UDP N-Acetylglucosamine 2epimerase, J Bacteriol 186, 706-712. [19] Vann, W. F., and Freese, S.J. (1994) Purification of Escherichia coli K antigens, Meth. Enzymol. 235, 304-311. [20] Morley, T. J., and Withers, S. G. (2010) Chemoenzymatic Synthesis and Enzymatic Analysis of 8Modified Cytidine Monophosphate-Sialic Acid and Sialyl Lactose Derivatives, Journal of the American Chemical Society 132, 9430-9437. [21] Mosley, S. L., Rancy, P. C., Peterson, D. C., Vionnet, J., Saksena, R., and Vann, W. F. (2010) Chemoenzymatic synthesis of conjugatable oligosialic acids, Biocatal Biotransfor 28, 41-50. [22] Wakarchuk, W., Martin, A., Jennings, M. P., Moxon, E. R., and Richards, J. C. (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis, J. Biol. Chem. 271, 19166-19173. [23] Gosselin, S., Alhussaini, M., Streiff, M. B., Takabayashi, K., and Palcic, M. M. (1994) A Continuous Spectrophotometric Assay for Glycosyltransferases, Anal. Biochem. 220, 92-97. [24] Muindi, K. M., McCarthy, P. C., Wang, T., Vionnet, J., Battistel, M., Jankowska, E., and Vann, W. F. (2014) Characterization of the meningococcal serogroup X capsule N-acetylglucosamine-1phosphotransferase, Glycobiology 24, 139-149. [25] Cho, J.-W., and Troy, F. A. (1994) Polysialic acid engineering: Synthesis of polysialylated neoglycosphingolipids by using polysialyltransferase from neuroinvasive Escherichia coli K1, Proc. Natl. Acad. Sci. U. S. A. 91, 11427-11431. [26] Steenbergen, S. M., and Vimr, E. R. (1990) Mechanism of polysialic acid chain elongation in Escherichia coli K1, Mol. Microbiol. 4, 603-611. [27] Ferrero, M. A., Luengo, J. M., and Reglero, A. (1991) H.p.l.c. of oligo(sialic acids): Application to the determination of the minimum chain length serving as exogenous acceptor in the enzymic synthesis of colominic acid, Biochem. J. 280, 575-579. [28] Willis, L. M., Gilbert, M., Karwaski, M. F., Blanchard, M. C., and Wakarchuk, W. W. (2008) Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme, Glycobiology 18, 177-186. [29] May, J. F., Splain, R. A., Brotschi, C., and Kiessling, L. L. (2009) A tethering mechanism for length control in a processive carbohydrate polymerization, Proc. Natl. Acad. Sci. U. S. A. 106, 1185111856. [30] Vijay, I. K., and Troy, F. A. (1975) Properties of membrane-associated sialyltransferase of Escherichia coli, J. Biol. Chem. 250, 164-170. [31] Lindhout, T., Bainbridge, C. R., Costain, W. J., Gilbert, M., and Wakarchuk, W. W. (2013) Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases, PLoS One 8, e69888.

40

Protein Accession Numbers:

41 42

1. Neisseria meningitidis group C alpha 2,9 polysialyltransferase UNIPROTKB : O06435

Genebank Accession: AAB53842

43 44

2. bovine liver nucleotide monophosphate kinase

UniProt Accession: Q2KIW9


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Biochemistry

1


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NmC PST mode of catalysis shifts from distributive to processive with increased acceptor chain length. 177x127mm (150 x 150 DPI)


Page 28 of 28

Effect of Acceptor Chain Length and Hydrophobicity on Polymerization

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