A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas

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A pseudo-isostructural type II DAH7PS enzyme from Pseudomonas aeruginosa: alternative evolutionary strategies to control shikimate pathway flux Oliver Sterritt, Sarah A Kessans, Geoffrey B. Jameson, and Emily J. Parker Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00082 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Biochemistry

A pseudo-isostructural type II DAH7PS enzyme from Pseudomonas aeruginosa: alternative evolutionary strategies to control shikimate pathway flux Oliver W. Sterritt1,2, Sarah A. Kessans1, Geoffrey B. Jameson1,2,3,* and Emily J. Parker1,2,4,* 1

Biomolecular Interaction Centre and School of Physical and Chemical Sciences, University of Canterbury, Christchurch, New Zealand

2

Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand 3

Institute of Fundamental Sciences and the Riddet Institute, Massey University, Palmerston North, New Zealand 4

Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand

Running title: Alternative evolutionary strategies to control shikimate pathway flux KEYWORDS: Shikimate pathway; 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; Pseudomonas aeruginosa PAO1; Type II DAH7PS

ACS Paragon Plus Environment

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ABSTRACT

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified. Here, the structure of a type II DAH7PS enzyme from P. aeruginosa (PAO1) has been determined at 1.54 Å resolution, in complex with its allosteric inhibitor tryptophan. Structural differences in the extra-barrel elements, when compared to other type II DAH7PS enzymes, directly relate to the formation of a distinct quaternary conformation with consequences for allosteric function and the control of flux to branching pathways. In contrast to the well-characterized Mycobacterium tuberculosis type II DAH7PS, which binds multiple allosteric inhibitors, this PaeDAH7PSPA2843 is observed to be modestly allosterically inhibited by a single aromatic amino acid, tryptophan. In addition, structures in complex with tyrosine or with no allosteric ligand bound were determined. These structures provide new insight into the linkages between the active and allosteric sites. With four putative DAH7PS enzymes, P. aeruginosa appears to have evolved control of shikimate pathway flux at the genetic level, rather than control by multiple allosteric effectors to a single type II DAH7PS, as in Mycobacterium tuberculosis. Type II DAH7PSs, thus, appear to have a more varied evolutionary trajectory than previously indicated.


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Biochemistry

INTRODUCTION As the first committed step of the shikimate pathway, the enzyme 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAH7PS) catalyzes the aldol-like condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form 3-deoxy-Darabino-heptulosonate 7-phosphate (DAH7P). Present in plants and microorganisms,1 but absent in mammals, the shikimate pathway leads to the biosynthesis of key aromatic metabolites, including the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)2 and, in the case of Pseudomonas aeruginosa, the toxic secondary metabolite pyocyanin.3

Based on enzyme sequence and sequence length, DAH7PSs are classified into three broad groupings: type Iα, type Iβ, or type II. Type I enzymes are smaller compared to their type II counterparts and there is less than 10 % sequence identity between the type I and type II classes. Despite this low sequence identity, all DAH7PS enzymes characterized to date share a common (βα)8 TIM barrel catalytic domain,4-14 with extra-barrel elements that are related to allosteric function, and to the formation of different quaternary assemblies. For example, Escherichia coli possesses three distinct type Iα DAH7PSs that each have an extension to loop α5β6 and an Nterminal barrel extension providing a single allosteric binding site that is selective for either Phe, Tyr, or Trp,4,15 whereas Thermotoga maritima possesses a single type Iβ enzyme that contains an N-terminal ACT domain6,16 that is associated with providing a single binding site for Tyr. In comparison, Mycobacterium tuberculosis expresses a single type II DAH7PS enzyme (MtuDAH7PS) that has both an N-terminal extension (providing structural elements β0, α0a, α0b, and α0c) and an extension to loop α2β3, providing helices α2a and α2b. These extra-barrel elements provide three distinct binding sites, on the single enzyme, that are selective for either


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Phe, Tyr, or Trp.8,17,18 The Phe and Trp sites, located at the dimer and tetramer interfaces respectively, are created upon formation of the quaternary assembly and contribute towards the complex synergistic allosteric control of MtuDAH7PS by binary or ternary combinations of aromatic amino acids involving Trp.17-19 The complex and sophisticated allosteric functionality of MtuDAH7PS is further extended by the formation of a non-covalent heterooctameric complex with chorismate mutase (MtuCM).20, 21 The formation of this complex not only activates MtuCM activity by more than two orders of magnitude but also allows MtuCM to access and utilize the allosteric machinery located on MtuDAH7PS to direct the shikimate pathway end product, chorismate, towards either Trp or Phe/Tyr biosynthesis according to precise metabolic requirements.20, 21

Although several examples of type I DAH7PSs have been characterized, both structurally and functionally, relatively limited structural information for the type II DAH7PSs has been reported. The structure of MtuDAH7PS (PDB 2B7O),8 along with complexes with aromatic amino acids (PDB 3NUE, 3NUD, and 2YPP)17,18 and in complex with MtuCM (PDB 2W19)20 and very recently a structure of the similar type II DAH7PS enzyme from Corynebacterium glutamicum (CglDAH7PS) in complex with Trp (PDB 5HUE) at 2.65 Å14 or in complex with CglCM (PDB 5HUD) are the only two examples of structurally characterized type II DAH7PS enzymes to date.

P. aeruginosa is an opportunistic human pathogen often associated with the chronic infection of patients suffering from cystic fibrosis.22 The DAH7PSs from P. aeruginosa are of particular interest as open reading frames that encode four putative DAH7PS isoenzymes, two that are


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Biochemistry

classified as type Iα and two that are classified as type II, have been identified. The two type II DAH7PS isoenzymes are encoded by the open reading frames PA2843 (PaeDAH7PSPA2843) or PA1901 (PaeDAH7PSPA1901).23 Sequence alignment among the type II enzymes from M. tuberculosis, C. glutamicum and P. aeruginosa (Figure S1) reveals that PaeDAH7PSPA2843 highly resembles MtuDAH7PS (50 % sequence identity, 67 % sequence similarity) and is predicted to contain similar accessory elements to those that are associated with allosteric function and the formation of quaternary structure in MtuDAH7PS. In comparison, PaeDAH7PSPA1901 is 43 amino acid residues shorter in sequence length, relative to PaeDAH7PSPA2843, and seems to lack much of the sequence that corresponds to the inserted helices α2a and α2b for MtuDAH7PS. PaeDAH7PSPA1901 is found in a gene cluster associated with the biosynthesis of the secondary metabolite pyocyanin and the absence of the inserted helices may have implications for the allosteric function and quaternary structure of this enzyme.

Here we report structures of the type II PaeDAH7PSPA2843 in complex with either Trp or Tyr, or with no allosteric ligand bound. The observed allosteric behavior relates directly to significant structural differences at regions previously associated with ligand binding in MtuDAH7PS, providing new insight into the linkages between the active and allosteric sites. These new structures illuminate the role of the extra-barrel elements in the formation of distinct quaternary assemblies, which are associated with unique allosteric properties of these type II enzymes, and cast light on the variety of allosteric inhibitory strategies adopted by the type II DAH7PSs indicating a more complex evolutionary trajectory for the type II DAH7PSs than data previously supported.


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EXPERIMENTAL METHODS

Protein expression and purification The open reading frame encoding PaeDAH7PSPA2843 was amplified from P. aeruginosa PAO1 gDNA using the polymerase chain reaction. The resultant PCR product was cloned into the expression vector pET-28a(+) engineered to incorporate an N-terminal tobacco etch virus (TEV) protease cleavable His6 purification tag. The complete plasmid was sequence verified (Macrogen), transformed into E. coli BL21*(DE3) cells and expression was achieved following the addition of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and subsequent incubation at 23 °C, with 200 rpm shaking, for 16 h. Cells were harvested by centrifugation (12,000 g, 15 min).

Cell lysis was achieved in lysis buffer [10 mM bis-Tris propane pH 8.0, 200 mM KCl, 1 mM tris(2-carboxyethyl)phosphine hydrochloride, 200 µM phosphoenolpyruvate, 20 mM imidazole] by sonication (4 x 5 min cycles at 80 % power). Cellular DNA was degraded by the addition of benzonase before the removal of debris by centrifugation (40,000 g, 30 min).

Purification was carried out using Ni2+ affinity chromatography, incubation with TEV protease (4 °C, 3 h), and size exclusion chromatography. Purified protein samples were flash frozen in liquid nitrogen and stored at -80 °C.

Primers were designed to incorporate the Leu179Asp substitution using the pET-28a(+) wild type

plasmid

as

a

template

for

PCR.

The

mutagenic

primers

were

Fwd:

5’-


6

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Biochemistry

GGCTTCGCCGACGACCACCAGGTGCAC-3’

and

Rev:

5’-

GTGCACCTGGTGGTCGTCGGCGAAGCC-3’. The mutagenic base pairs responsible for the substitution are highlighted in bold. The resultant plasmids were sequence verified (Macrogen) and expression and purification of the mutant PaeDAH7PSPA2843 Leu179Asp was carried out as described above for the wild type enzyme.

Mass spectrometry The molecular weight of PaeDAH7PSPA2843 was determined by electrospray ionization mass spectrometry (Bruker maXis 3G). Protein samples were dialyzed into Milli-Q water and diluted to a concentration of 1 mg/mL prior to analysis. The molecular mass of a single chain of PaeDAH7PSPA2843 was found to be 49,990 Da compared to the calculated theoretical mass of 49,992 Da (ProtParam).

Enzyme kinetic assays The activity of PaeDAH7PSPA2843 was monitored over a range of pHs from pH 6.0 – pH 8.5 and over a range of temperatures from 35 °C – 40 °C based on methods previously described8 by monitoring the consumption of PEP at 232 nm using a Varian Cary 300 UV-Vis spectrophotometer. Metal ion preference was investigated by monitoring the activity of PaeDAH7PSPA2843 in the presence of various divalent metal cations. The enzyme was pretreated with 0.5 mM EDTA for 2 hrs to remove background metal ions before being buffer exchanged into assay buffer pretreated with Chelex (Bio-Rad). The appropriate test metal was added at 100 µM. PEP (Sigma) and E4P (Sigma) concentrations were held at 150 µM, except when determining the respective KM value, which was determined by monitoring activity in the


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presence of 10 – 600 µM of the substrate for which KM was being determined. All reactions were carried out in the presence of 100 µM Co2+ (except when determining metal ion preference). The reaction was initiated by the addition of purified PaeDAH7PSPA2843 and initial reaction rates were determined using a least-squares fit of the data.

Differential Scanning Fluorimetry The thermal stability of PaeDAH7PSPA2843 was measured using an applied Biosystems QuantStudio 3. Samples were prepared in buffer, pH 7.5, and contained the appropriate aromatic amino acid, SYPRO Orange fluorescent dye, and a final enzyme concentration of 0.6 mg/mL. The plate was sealed and heated from 20 °C to 90 °C. Samples were measured in triplicate and negative controls, where protein was substituted by buffer were run in parallel. Melting temperatures were determined by the inflection point of the melt curve, as defined by the maximum derivative.

Isothermal titration calorimetry Isothermal titration calorimetry (ITC) experiments were carried out using a TA Nano ITC instrument. ITC measurements were carried out in buffer containing 50 mM bis-Tris propane pH 7.5, 200 µM phosphoenol pyruvate at 298 K, with 200 rpm stirring. All solutions were degassed immediately prior to use. The ligand was titrated over a series of 20 injections (1 x 1.0 µL and 19 x 2.5 µL) with the first data point removed to allow for the diffusion of ligand during the equilibration. Heats of dilution measurements were made and subtracted from the integrated experimental data before curve fitting using Nanoanalyze software.


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Biochemistry

Analytical ultracentrifugation Protein samples were dialyzed against buffer containing 50 mM bis-Tris propane pH 7.5, 200 mM KCl, 100 µM cobalt chloride, 200 µM phosphoenol pyruvate at 4 °C for 2-3 h.

Analytical ultracentrifugation sedimentation velocity experiments were conducted in a Beckman-Coulter model XL-I instrument set at a temperature of 20 °C. Protein samples were diluted in buffer (50 mM bis-Tris propane pH 7.5, 200 mM KCl, 100 µM cobalt chloride, 200 µM phosphoenol pyruvate) to establish protein samples at three concentrations (1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL). For each protein sample, 380 µL of protein and 400 µL of reference buffer (50 mM bis-Tris propane pH 7.5, 200 mM KCl, 100 µM cobalt chloride, 200 µM phosphoenol pyruvate) were loaded into double-sector quartz cells. Cells were mounted in a Beckman-Coulter 8-hole An-50 Ti rotor and centrifuged at a rotor speed of 35,000 rpm. Data were collected at a wavelength of 280 nm, in continuous mode for 200 scans, using a time interval of 0 s, and a step-size of 0.003 cm without averaging. Solvent density (1.0129 g/mL) and viscosity (1.050 cP) were measured using an Anton Paar Lovis DMA4100M density meter or an Anton Paar Lovis 2000 ME microviscometer respectively, and partial the specific volume of the protein (0.73049 mL/g) was calculated using the amino acid composition in the program SEDNTERP. Sedimentation velocity data at multiple time points were fitted to a continuous sedimentation-coefficient [c(s)] model24-26 using the program SEDFIT.

Small angle X-ray scattering data collection and analysis


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SEC-SAXS data were collected at the SAXS/WAXS beamline at the Australian Synchrotron27 using a sheath flow sample environment28 at 12 keV (1.0332 Å), with a detector distance of 1600 mm, and at a temperature of 293 K. Data were collected using a SEC-input protein concentration of 5 mg/mL in buffer containing 50 mM bis-Tris propane pH 7.5, 100 µM cobalt chloride, 200 µM phosphoenol pyruvate, 5 % glycerol, in the presence or absence of 1 mM Trp.

Data were processed using the reduction software ScatterBrain 2.83, developed at the Australian Synchrotron. Scattering intensity (I) was plotted versus q, as a log-linear plot, and analyzed using the ATSAS package.29 Theoretical scattering data were calculated from the tetrameric crystal structures of PaeDAH7PSPA2843 or MtuDAH7PS and compared to the experimental scattering data using CRYSOL.30

Crystallography and structure determination Protein crystals were prepared by mixing equal volumes of purified protein (final protein concentration 7 – 8 mg/mL) with reservoir solution (0.1 M sodium acetate (pH 5.5), 0.8 M sodium formate, 1 mM cobalt chloride, 1 mM phosphoenol pyruvate, 26 % PEG 2000 MME) and incubated at 278 K for 4 – 7 days. Crystals containing allosteric ligands were obtained by the addition of either 2 mM Trp or 3 mM Tyr to the crystallization reservoir prior to the addition of protein.

Crystals were flash frozen at 110 K in cryoprotectant containing 25 % PEG 300 and mother liquor. X-ray diffraction data were collected at the Australian Synchrotron using the MX beamlines31 at a wavelength of 0.9537 Å with data collection every 0.5 ° using a Quantum 210r


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Biochemistry

(ADSC) CCD detector. For the Tyr-bound and allosteric ligand-free data sets, the detector distance was set to minimize reflection overlaps thus limiting the resolution available for refinement.

Structures were solved by molecular replacement (MOLREP)32 using a single chain of MtuDAH7PS8 (PDB 2B7O) as a search model. All ligands and waters were removed from the search model prior to molecular replacement. Models were built using COOT33 and refined with REFMAC.34

Interface analysis PISA35 was used to visualize and examine the residues involved in interface formation. LSQKAB36 was used to superpose and compare the structures. PyMol was used to visualize all structures.

Protein Data Bank accession codes Atomic coordinates and structure factors for the structures described in this work have been deposited in the Protein Data Bank with the accession codes 5UXO (allosteric ligand free), 5UXN (Tyr bound) and 5UXM (Trp bound).


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RESULTS AND DISCUSSION

PaeDAH7PSPA2843 is selectively inhibited by Trp The open reading frame encoding PaeDAH7PSPA2843 was amplified from P. aeruginosa PAO1 gDNA and cloned into the expression vector pET28a(+). The recombinant protein was overexpressed and purified. The enzyme was found to be catalytically active with maximal activity observed at pH 7.5, 37 °C. Metal ion preference was investigated by monitoring the activity of PaeDAH7PSPA2843 in the presence of various divalent metal cations (Figure 1) and it was found that Co2+ was most activating, as is the case for other type II DAH7PSs,8,37 and as such was used in all subsequent functional assays. Apparent KM values for PEP and E4P were determined to be 28 ± 1 µM and 18 ± 1 µM respectively. Although the Michaelis constants are in line with other characterized type II DAH7PS enzymes.8,37,38 the turnover number, kcat, was found to be an order of magnitude higher (kcat = 40 ± 0.7 s-1).

Table 1: Kinetic parameters determined for PaeDAH7PSPA2843. Enzyme

PaeDAH7PSPA2843

Metal Ion Co2+

KmE4P

KmPEP

kcat

kcat/ KmE4P

kcat/ KmPEP

(µM)

(µM)

(s-1)

(s-1 µM-1)

(s-1 µM-1)

18 ± 1

28 ± 1

40.0 ± 0.7

2.20 ± 0.20

1.40 ± 0.10

The activity of PaeDAH7PSPA2843 was monitored in the presence of increasing concentrations of the aromatic amino acids Phe, Tyr and Trp. At a concentration of up to 200 µM Phe or Tyr, minimal change in enzymatic activity was observed. However, in the presence of up to 200 µM


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Biochemistry

Trp, a decrease in catalytic activity was evident with a reduction in activity to ~75 % of that in the absence of Trp (Figure 1). Combinations of aromatic amino acids, which have a dramatic effect on the activity of MtuDAH7PS,17,18 appear to have no additional inhibitory effect, with inhibition levels similar to that of Trp alone when Trp was present. This behavior is in distinct contrast to that observed for MtuDAH7PS where any binary or ternary combination of aromatic amino acids that includes Trp has been shown to synergistically inhibit the enzyme.17,18,39

To ascertain whether the enzyme was capable of binding aromatic amino acids, isothermal titration calorimetry experiments were carried out (Figure S2). Titration of Trp yielded a dissociation constant (Kd) of 3.6 ± 0.8 µM (n = 0.92), whereas Tyr was observed to bind an order of magnitude less tightly with Kd = 23 ± 1 µM (n = 1.07).

Differential scanning fluorimetry experiments show that in the presence of 200 µM Trp an increase in thermal stability of 6.1 ± 0.2 °C is evident, whereas in the presence of 200 µM Phe or Tyr, changes in thermal stability were not observed which may correlate with the lower levels of inhibition seen in the presence of Phe or Tyr relative to that seen in the presence of Trp. Binary or ternary combinations of aromatic amino acids with Trp present produced an increase in thermal stability similar to that observed for Trp alone.


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Figure 1. Activity of PaeDAH7PSPA2843 A: In the presence of various divalent metal cations. B: In the presence of single aromatic amino acids and C: In the presence of binary or ternary combinations of aromatic amino acids (each single letter code represents 200 µM of the corresponding aromatic amino acid). Assays were carried out in the presence of 150 µM of each of PEP and E4P. D: Thermal stability of PaeDAH7PSPA2843 in the presence or absence of combinations of aromatic amino acids. Each single letter code represents 200 µM on the


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Biochemistry

corresponding amino acid. For all figures, error bars represent the standard deviation of triplicate measurements.

The crystal structure of PaeDAH7PSPA2843 Three crystal structures of PaeDAH7PSPA2843 were solved in complex with either Trp (1.54 Å, Rfree = 0.210), Tyr (2.2 Å, Rfree = 0.225), or with no allosteric ligand bound (2.35 Å, Rfree = 0.259). For all structures, PaeDAH7PSPA2843 crystallized in the space group I222, with one DAH7PS chain (residues 4 – 446) present in the asymmetric unit. Application of two two-fold symmetry operations generates two significant interfaces between neighboring asymmetric units, assembling a homotetramer (Figure 2). Data collection and refinement statistics are shown in Table 2.

Table 2. Data collection and refinement statistics for the three crystal structures determined for PaeDAH7PSPA2843. Allosteric ligand Trp-bound free

Tyr-bound

PDB Code

5UXO

5UXM

5UXN

Space Group

I222

I222

I222

a

80.24

81.68

81.27

b

100.12

100.37

100.54

c

115.60

115.60

115.55

Data collection and processing statistics Unit Cell Dimensions (Å)


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Resolution range (outer shell) 57.80-2.35 (Å) (2.43-2.35)

57.80-1.54 (1.57-1.54)

57.63-2.20 (2.27-2.20)

Wavelength (Å)

0.9537

0.9537

0.9537

Total measurements

139,908 (13,875)

491,613 (24,675)

173,797 (15,185)

Unique reflections

19,777 (1,924)

70,122 (3,437)

24,329 (2,093)

Completeness (%)

100.00 (100.00)

99.60 (99.80)

100.00 (100.00)

Redundancy

7.1 (7.2)

7.0 (7.2)

7.1 (7.3)

I/σ

9.9 (4.2)

9.3 (1.5)

10.5 (6.4)

Rmerge

0.139 (0.392)

0.106 (1.122)

0.139 (0.326)

Rpim

0.056 (0.156)

0.044 (0.452)

0.056 (0.130)

CC1/2

0.992 (0.962)

0.995 (0.702)

0.992 (0.936)

Refinement resolution (Å)

2.35 (2.43-2.35)

1.54 (1.57-1.54)

2.20 (2.27-2.20)

R factor

0.204

0.185

0.184

Rfree (5 %)

0.259

0.210

0.225

Protein atoms

3382

3566

3452

Water atoms

126

340

181

Ligand atoms

20

38

35

Wilson B value (Å2)

14.1

13.2

13.3

All atoms

33.0

20.1

23.9

Protein atoms

33.4

20.2

24.8

Ligand atoms

29.3

20.8

22.1

Water atoms

28.2

28.9

23.3

Refinement statistics

Number of non-hydrogen atoms

Mean B value (Å2)

Root mean square-deviations from ideality


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Biochemistry

Bond lengths (Å)

0.015

0.010

0.023

Bond angles (degrees)

1.9

1.4

1.6

Residues in the most favored 99.0 region of Ramachandran plot (%)

98.0

97.0

Residues in forbidden region of 0.00 Ramachandran plot (%)

0.25a

0.23b

a

Leu218

b

Thr421

Figure 2. The structure of PaeDAH7PSPA2843 (PDB 5UXM) in complex with the allosteric inhibitor Trp. A: A single chain of PaeDAH7PSPA2843. The (βα)8 catalytic domain is shown in blue, the N-terminal extension (residues 1-59) is shown in red and the extension to loop α2β3 (residues 179-225) is shown in yellow. The active-site metal ion is indicated in magenta and the allosteric inhibitor Trp is shown in green. B: The more extensive dimer interface (indicated by


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black dashed line) is formed, in part, by helix α2 whereas the less extensive tetramer interface (indicated by cyan dashed line) is formed, in part, by helices α2a and α2b.

The final model for the Trp-bound structure (PDB 5UXM) contains one Trp ligand with very well defined electron density, the Tyr-bound structure (PDB 5UXN) contains one Tyr ligand, and the allosteric ligand free structure (PDB 5UXO) contains one acetate ion bound at the allosteric binding site (Figure 3). The B-factors for the Trp and Tyr ligands are similar to the Bfactors of neighboring protein residues, indicating full occupancy of the ligand. All three structures contain one well defined PEP molecule modeled into the active site of the enzyme along with one Co2+ ion, with attached water molecule, observed at partial occupancy (~0.8) (Figure 3). From the electron density attributed to the PEP ligand, the methylene carbon C3 and the carboxylate group of PEP are coplanar. Moreover, the catalytically important nucleophilic water that is hydrogen-bonded to the side chain of the absolutely conserved Glu234 (PaeDAH7PSPA2843 numbering) hovers only 3.0 Å from the olefin of PEP.40 This PEP binding mode in PaeDAH7PSPA2843 is unlike that modelled for the MtuDAH7PS or CglDAH7PS structures, where the methylene carbon C3 and the carboxylate group of PEP are not coplanar, although it is noted that these structures are at lower resolutions and differences in the modeled PEP conformation may only be of marginal significance. However, this planar PEP conformation may account for the altered reactivity observed for PaeDAH7PSPA2843.


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Figure 3: The active site and allosteric ligand-binding site of PaeDAH7PSPA2843. A: The binding mode of the substrate PEP (PDB 5UXM). The active-site metal ion, Co2+, is shown in magenta, the phosphate ion, indicating the likely location of the E4P phosphate group, is shown in orange and the nucleophilic water is shown in red. B: The binding mode of the allosteric inhibitor Trp (PDB 5UXM). C: Overlay between the Trp binding sites of PaeDAH7PSPA2843 (PDB 5UXM) MtuDAH7PS (PDB 3NUE) and CglDAH7PS (PDB 5HUE) shows similar Trp binding modes in the three enzymes. PaeDAH7PSPA2843 is shown in green, MtuDAH7PS is shown in cyan, and CglDAH7PS is shown in magenta. D: The binding mode of Tyr, bound at the Trp site, in PaeDAH7PSPA2843 (PDB 5UXN). For all figures, the black mesh represents 2|Fo|-


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|Fc| electron density map contoured at 1 σ. Residue numberings correspond to PaeDAH7PSPA2843, * indicates residue numbering corresponding to MtuDAH7PS, and ^ indicates residue numbering corresponding to CglDAH7PS.

As with all DAH7PS structures, each chain of PaeDAH7PSPA2843 features a core (βα)8 triose phosphate isomerase fold, with extensions to the barrel that place it in the type II family (Figure 2). Residues 1 – 59 form an N-terminal extension to the core (βα)8-barrel catalytic domain, providing an additional three helices (α0a, α0b, and α0c). Residues 179 – 225 form an extension to loop α2β3, providing a 1-turn helix, α2a, and a 6-turn helix, α2b.

The dimer interface is formed through interactions between chains A and B (and chains C and D), in particular helices α0c and α2. A total of 40 residues from each chain are involved in the formation of this interface, which is comprised of five residues involved in hydrogen bond formation and a buried interface area of 1202 Å2 (7.0 % of the surface area of each chain; ∆G = 8.3 kcal/mol). Three pairs of equivalent hydrogen bonds are formed between chain A atoms Leu40_O, Thr162_OG1, Arg169_NE and chain B atoms Asn166_ND2, Thr162_OG1, Asn166_OD1 respectively. In addition, Glu226_OE1 shares a proton with its crystallographic equivalent across the dimer interface. Glu226 may adopt an alternative, non-interacting, conformation at pH 7.5 (the pH at which solution experiments were carried out). However, this dimer interface is predominantly formed by hydrophobic interactions and the effect of the difference in pH between the crystallization condition and solution experiments on the


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Biochemistry

quaternary structure is likely negligible. Upon binding of the allosteric ligand Trp, there are no significant changes to hydrogen-bonding arrangements at the dimer interface.

The tetramer interface is formed through interaction between chains A and D (or chains B and C) involving residues primarily located in helices α2a and α2b. A total of 24 residues and a buried interface area of 915 Å2 (5.3 % for each chain; ∆G = -9.7 kcal/mol) contribute towards the formation of the tetramer interface. Five residues are involved in the formation of hydrogen bonds and two residues are involved in the formation of salt bridges across the tetramer interface. For the allosteric ligand free structure (PDB 5UXO), hydrogen bonds are formed between chain A atoms Arg205_NH1, Glu208_OE1, Thr209_OG1 of helix α2b and chain D atoms Glu208_OE1, Arg205_NH1, Thr209_OG1 of helix α2b respectively. On binding Trp, these contact distances all increase, especially the Thr209_OG1...OG1_Thr209 contact, which increases by more than 0.7 Å.

Comparison between type II structures reveals distinct quaternary assemblies Alignment of the single chain of PaeDAH7PSPA2843 (PDB 5UXO) and one or other of the two crystallographically independent chains of MtuDAH7PS (PDB 2B7O, here and below) (rmsd = 1.27 and 1.32 Å, Table S1) shows a high degree of structural similarity between the monomeric units. Notable differences between the two structures exist at helices α2a and α2b as well as in the N-terminal regions. In MtuDAH7PS, helix α2a is a 3-turn helix (residues 194 – 207) whereas in PaeDAH7PSPA2843 this helix is of only 1-turn (residues 182 – 186). Helix α2b is a 5-turn helix (residues 211 – 231) in MtuDAH7PS compared to the 6-turn helix (residues 193 – 216) observed


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in PaeDAH7PSPA2843. Further to this, an additional 18 residues at the N-terminus of MtuDAH7PS form β strand β0 and loop β0α0a, which are not observed in PaeDAH7PSPA2843.

Alignment between the dimeric DAH7PS units of PaeDAH7PSPA2843 and MtuDAH7PS (rmsd = 1.56 Å for the dimer interface and 5.74 Å for the tetramer interface) reveals distinct differences between the dimer interfaces of both structures. Notably, the N-terminal region β0 (residues 1 – 12) in MtuDAH7PS forms a two stranded anti-parallel β sheet across the dimer interface which contributes towards a more substantial dimer interface in MtuDAH7PS relative to the dimer interface seen in PaeDAH7PSPA2843, with a buried interface area of 1856 Å2 (9.5 %) and 1202 Å2 (7.0 %) of the surface of each chain respectively. Nonetheless these dimer units share a very similar quaternary structure, as the rmsds for monomer subunits are only slightly smaller than that for the dimer.

Comparison between the tetrameric species of PaeDAH7PSPA2843 (PDB 5UXO, 5UXM, or 5UXN) and MtuDAH7PS (PDB 2B7O) (rmsd = 3.08, 3.36 or 4.43 Å respectively) reveals the formation of a significantly different tetramer assembly in PaeDAH7PSPA2843 relative to either MtuDAH7PS or CglDAH7PS. Although the extent of the tetramer interface is comparable between these enzymes, differences in the angle of intersect between helices α2b from each of chains A and D contribute towards the formation of two different quaternary structures. In MtuDAH7PS or CglDAH7PS, helices α2b intersect across the tetramer interface at a relative angle of ~60 ° whereas in PaeDAH7PSPA2843 these helices intersect at a relative angle of ~90 ° (Figure 4). This difference brings residues Arg205 (chain A) and Glu208 (chain D) into an orientation favorable for the formation of a salt bridge across the tetramer interface in


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Biochemistry

PaeDAH7PSPA2843. This, in combination with the differences seen at the dimer interface, contributes towards a pronounced tetrahedral twisting across the tetramer interface in PaeDAH7PSPA2843 resulting in a significant relative displacement (~10 Å) of the active site Co2+ ion in chains C and D when compared to the position of the active site Mn2+ ion in MtuDAH7PS or CglDAH7PS, when the conserved dimers are superposed, despite the conservation of the metal binding position in a single chain of these enzymes. This pronounced twisting leads to a PaeDAH7PSPA2843 tetramer that adopts a tetrahedral conformation in comparison to the nearly planar tetramer conformation observed for MtuDAH7PS or CglDAH7PS. This distinct difference in the overall homotetrameric conformation may also mirror a difference in function. Both the MtuDAH7PS and CglDAH7PS form noncovalent associations with the AroQδ subclass chorismate mutase (MtuCM or CglCM respectively). The P. aeruginosa genome encodes two CMs, corresponding to open reading frames PA3166 (PaeCMPA3166) and the PA5184 (PaeCMPA5184). PaeCMPA3166 is part of a bifunctional P-protein containing both CM and prephenate dehydratase domains, and this CM is of AroQα subclass. PaeCMPA5184 shows high similarity to the AroQγ subclass, which are a group of secreted CMs,41 and is hence unable to participate in the intracellular biosynthesis of aromatic amino acids. P. aeruginosa thus does not contain an AroQδ subclass CM, which has been shown to form interactions across the tetramer interface of both MtuDAH7PS42 and more recently CglDAH7PS.14


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Figure 4. Comparison between the orientation of tetramer interface helix α2b in PaeDAH7PSPA2843 (PDB 5UXM) relative to MtuDAH7PS (PDB 2B7O) or CglDAH7PS (PDB 5HUE). PaeDAH7PSPA2843 is shown in blue with helix α2b highlighted in cyan. MtuDAH7PS is shown in green, with helix α2b highlighted in magenta. CglDAH7PS is shown in grey.


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Biochemistry

Crystal structures of PaeDAH7PSPA2843 reveal a single aromatic amino acid allosteric binding site When co-crystallized with either 2 mM Trp or 3 mM Tyr, Trp or Tyr were found to bind at a similar allosteric binding site (Figure 3) despite observed differences in allosteric sensitivity and enzyme thermal stability when Trp is bound compared to when Tyr is bound (Figure 1). The allosteric binding site is located at the tetramer interface, approximately 26 Å from the active site and is formed between helices α1 and α2a of chain A and helix α2b of chain D. A combination of 15 residues from chain A and 3 residues from chain D, with 156 Å2 (0.9 %) and 52 Å2 (0.7 %) of surface area of each chain respectively involved in interacting with the Trp ligand. Hydrogen bonds are formed between chain A atoms Ala177_O, Gln223_O and Glu226_O and atoms NE1, N, and N of the Trp ligand respectively. Two stabilizing salt bridges are formed between chain A atom Lys105_NZ and atoms O and OXT of the Trp ligand. A pi-cation interaction occurs between Lys105 and the Trp indole ring. In addition, hydrophobic interactions occur between the indole ring of the Trp ligand and the side chains of residues Leu89, Val93, Leu179, Trp185 and Thr227_CB of chain A and residues Phe212 and Cys216 of chain D.

The single allosteric binding site observed in PaeDAH7PSPA2843 is comparable to the region associated with Trp binding in both MtuDAH7PS (PDB 3NUE)17 and CglDAH7PS (PDB 5HUE),14 both in terms of location and sequence similarity, although it is noted that the Trp ligand is poorly defined in CglDAH7PS (Figure 3). Subtle differences in the Trp binding mode exist among the three enzymes. Most notably, an extra hydrogen bond is formed in PaeDAH7PSPA2843 between the side chain hydroxyl group of Thr227 and atom OXT of the Trp


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ligand that is not seen in either MtuDAH7PS or CglDAH7PS due to the identity of the residue found in the equivalent position (Ala241 and Ala251 respectively).

Interestingly, we have observed in the structure of PaeDAH7PSPA2843 a second substantial cavity, located in close proximity to both the dimer and tetramer interfaces. In the presence of Trp, this pocket becomes considerably enlarged and elongated. The functional significance of this pocket is yet to be determined but the biosynthesis of pyocyanin from the end product of the shikimate pathway, chorismate, is noted (Figure S4).

For MtuDAH7PS, two further allosteric binding sites exist – one is found at the dimer interface and binds Phe, while the other is found in the N-terminal region and binds Tyr. Comparison between PaeDAH7PSPA2843 and these two further regions of MtuDAH7PS that are associated with either Phe or Tyr binding17,18 reveals significant structural differences that preclude the binding of either Phe or Tyr at equivalent sites in PaeDAH7PSPA2843 (Figure 5).


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Figure 5. Surface comparisons of the regions associated with Phe or Tyr binding in MtuDAH7PS (PDB 2YPO or 2YPP) and equivalent regions in PaeDAH7PSPA2843 (PDB 5UXM). A: β Strand β0 (light pink) contributes towards the formation of a deep Phe binding pocket in MtuDAH7PS (chain A shown in magenta, chain B shown in purple, Phe ligand shown in green). B: The less extensive N-terminal region found in PaeDAH7PSPA2843 leads to a shallow area that is unable to support Phe binding (chain A shown in cyan, chain B shown in blue, the equivalent MtuDAH7PS Phe region is indicated by the red square). C: The MtuDAH7PS Tyr binding region, formed, in part, by β strand β0 is found in the N-terminal region of the enzyme


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(chain A is shown in magenta, chain B shown in purple, and the Tyr ligand shown in green). D: The equivalent MtuDAH7PS Tyr binding region in PaeDAH7PSPA2843 is shallow due to a less extensive N-terminal region and is unable to support Tyr binding (chain A shown in cyan, chain B shown in blue, and the equivalent MtuDAH7PS Tyr binding region is indicated by the red square).

For the region associated with Phe binding in MtuDAH7PS, the shorter N-terminal extension found in PaeDAH7PSPA2843, in particular the absence of β strand β0, leads to a shallow area that is more exposed in PaeDAH7PSPA2843. Further to this, the side chains of residues Tyr37, Phe76, and His159 alter the size and shape of this pocket in PaeDAH7PSPA2843 and contribute towards preventing Phe binding in this region.

For the region associated with Tyr binding in MtuDAH7PS, the absence of β strand β0 in PaeDAH7PSPA2843 also leads to the formation of a shallow area in PaeDAH7PSPA2843 that is unable to support the binding of a Tyr ligand. Instead, Tyr is observed to bind PaeDAH7PSPA2843 at the same allosteric binding site compared to Trp. However, the Tyr ligand does not form a salt bridge interaction with Lys105 and five additional water molecules are also observed to occupy the allosteric binding pocket. Apart from these differences, there are no obvious structural differences between Trp-bound and Tyr-bound proteins at the allosteric site that would account for the observed functional differences of Trp versus Tyr binding. When the active sites of Trpbound, Tyr-bound, and allosteric ligand-free structures are compared, there are no clear structural differences that can account for the differences in activity of these proteins. Thus, it appears that, as is predicted for MtuDAH7PS,39 dynamic differences may drive the different


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Biochemistry

catalytic activities in the presence of Trp or Tyr and while these ligands may not lead to average conformational differences, they may alter dynamic signal communication between the active and allosteric sites; while Tyr can bind at the allosteric site, it would appear that it cannot activate the same dynamic communication network as Trp. In this regard, it should also be noted that there are differences in the thermodynamics of ligand binding; Trp binding is largely enthalpically favored (∆G = -31.0 kJ/mol, ∆H = -28.0 ± 1 kJ/mol, ∆S = 11.8 J/mol K) and Tyr binding is predominantly entropically favored (∆G = -26.5 kJ/mol, ∆H = -4.0 ± 1 kJ/mol, ∆S = 75.8 J/mol K). These differences may relate to the altered functional response to Tyr compared to Trp binding.

Site-directed mutagenesis was employed to confirm the presence of a single aromatic amino acid binding site in PaeDAH7PSPA2843. The Trp binding site mutant PaeDAH7PSPA2843 Leu179Asp was selected as a suitable mutation to disrupt the extensive stabilizing interactions made between Leu179 and the Trp ligand. The mutant enzyme PaeDAH7PSPA2843 Leu179Asp was found to be catalytically active with Michaelis constants (KM(PEP) = 37 ± 3 µM, KM(E4P) = 23 ± 3 µM, kcat = 26.6 ± 0.8 s-1) comparable to those determined for the wild-type enzyme. However, in the presence of up to 200 µM Trp, no significant loss in enzymatic activity was observed, in contrast to that observed for the wild-type enzyme (Figure S5).

Analytical ultracentrifugation and small-angle X-ray scattering data confirms tetrameric solution structure


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Sedimentation velocity experiments conducted at concentrations 0.25, 0.5, and 1.0 mg/mL, at pH 7.5, give a sedimentation coefficient of 9.1 S, corresponding to a calculated molecular weight of 202 kDa, consistent with a tetrameric species in solution (Figure. 6). The sedimentation coefficient of PaeDAH7PSPA2843 remained constant at all concentrations tested. While a minor peak at ~4S was observed, the majority of the enzyme was found to be predominantly in the tetrameric form, even at low enzyme concentrations.

Figure 6: Sedimentation velocity data collected for PaeDAH7PSPA2843 show the distribution of the solution-state species. Data were collected at 35,000 rpm at a temperature of 20 °C using protein concentrations of 0.25 (black), 0.50 (red) and 1.0 mg/mL (blue).

Size exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS) data, collected in the presence and absence of Trp, also supports a tetrameric species in solution (SAXS MW = 219.9 kDa). Comparison between the experimental scattering profiles obtained in the presence and absence of the allosteric inhibitor Trp (Figure. 7, Table 2) indicates that no


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Biochemistry

major change in enzyme shape is observable on Trp binding, although some changes in enzyme flexibility are evident, consistent with observations made in the single-crystal diffraction experiments. Comparison between SEC-SAXS data collected for the wild type enzyme and the Leu179Asp mutant indicates that this mutation did not cause any observable structural change (Figure. S6, Table S2).

SEC-SAXS data were also compared to the theoretical scattering profile calculated from either the tetrameric crystal structures of PaeDAH7PSPA2843 (PDB 5UXM, 5UXN, or 5UXO) or from the tetrameric crystal structure of MtuDAH7PS (PDB 2B7O, 3NUE or 5CKV). Theoretical scattering profiles were calculated using CRYSOL.30 The fits of the theoretical scattering profiles calculated from the tetrameric crystal structure of PaeDAH7PSPA2843 to the experimental data collected in the presence or absence of Trp (Figure. 8) indicate that the solution-state structure is similar to the quaternary assembly seen in the crystal structure. However, fits of the theoretical scattering profiles calculated from the tetrameric crystal structures of MtuDAH7PS (Figure. 8) are suggestive of some variation in overall shape between the two enzymes, consistent with observations seen in the single-crystal X-ray diffraction results.


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Figure 7. SEC-SAXS results for PaeDAH7PSPA2843 in the presence (blue circles) and absence (red squares) of the allosteric inhibitor Trp. A, log I(q) versus q, error bars are indicated in grey, with the theoretical scattering profile calculated from the tetrameric Trp bound structure (PDB 5UXM) overlaid (black line). B, Guinier plot (ln(I) versus q2). C, Kratky plot (q2⋅I(q) versus q) for the data in (A). D, P(r) versus r profiles for the data in (A). Where error bars are not visible, they are contained within the symbol.


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Biochemistry

Table 2. SAXS parameters determined for PaeDAH7PSPA2843 in the presence or absence of the allosteric inhibitor Trp. PaeDAH7PSPA2843

Ligand Free

+ Trp

Rg (Å)

42.38

40.47

I(0) (cm-1)

0.135 ± 0.001

0.130 ± 0.001

qmin (Å-1)

0.010

0.010

Correlation coefficient, r2

0.999

0.999

Rg (Å)

41.40 ± 0.6

41.0 ± 0.2

I(0) (cm-1)

0.130

0.130

dmax (Å)

125.0

133.0

Vp (Å3)

299,900

294,700

q range (Å-1)

0.010-0.258

0.010-0.258

MW (Da)

219,880

213,270

Number of subunits

4

4

Guinier Analysis

Pair Wise Distribution Analysis

MW Estimate (SAXS MoW)


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Figure 8. The CRYSOL fits of the theoretical scattering profiles calculated from the tetrameric crystal structures of either PaeDAH7PSPA2843 (PDB 5UXO, 5UXN, or 5UXM) or MtuDAH7PS (PDB 2B7O, 3NUE, or 5CKV) to the experimental SEC-SAXS data obtained


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Biochemistry

for PaeDAH7PSPA2843 in the presence (blue circles) or absence (red squares) of the allosteric inhibitor Trp. The calculated theoretical scattering profile for the corresponding tetrameric crystal structure is shown as a black line.

Evolutionary implications The structural commonalities shared by the structural elements inserted into the core (βα)8barrel of both PaeDAH7PSPA2843 and MtuDAH7PS suggests a common origin for these elements. Assuming that the acquisition of the extensions to the N-terminus was not an iterative, piecemeal event and that this extension was obtained by gene fusion events, it appears that PaeDAH7PSPA2843 may have lost the highly complex allosteric regulation that is seen in MtuDAH7PS. Whereas M tuberculosis has evolved a single DAH7PS under synergistic allosteric control by combinations of aromatic amino acids that include Trp, and shares its allosteric sites with MtuCM for more advanced pathway control, P. aeruginosa has taken an alternative evolutionary trajectory for controlling entry into the shikimate pathway. Given that PaeDAH7PSPA2843 is not the sole DAH7PS for P. aeruginosa, genetic control may be more appropriate; especially as the DAH7PSs in this case function to deliver primary metabolites to support protein synthesis as well as the secondary metabolite pyocyanin.


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CONCLUSIONS

The structure of PaeDAH7PSPA2843 reveals significant differences in the formation of quaternary structure within the type II class of DAH7PS enzymes, despite the high level of sequence similarity found between members of the class, and illuminates a variety of allosteric inhibitory strategies adopted by type II DAH7PSs.

Relative to MtuDAH7PS or CglDAH7PS, the PaeDAH7PSPA2843 tetramer has a substantial tetrahedral twist that is associated with the diminished allosteric function of PaeDAH7PSPA2843 when compared with that observed for MtuDAH7PS. The N-terminal residues that form the Phe or Tyr allosteric binding regions in MtuDAH7PS are absent in PaeDAH7PSPA2843 and subsequently PaeDAH7PSPA2843 has a single aromatic amino acid allosteric binding site that is selective for Trp.

The observed structural differences in the extra-barrel elements, relative to MtuDAH7PS, suggests that PaeDAH7PSPA2843 has lost the complex allosteric machinery that has evolved in MtuDAH7PS and indicates that P. aeruginosa relies upon an alternative strategy, at the gene transcription level, to regulate the flux of chorismate away from aromatic amino acid biosynthesis, indicating that the type II DAH7PSs have a more complex evolutionary trajectory than data have previously indicated.


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ASSOCIATED CONTENT Supporting Information The following file is available free of charge (PDF):

Figure S1. Sequence alignment of type II DAH7PS enzymes, indicating the relative positions of the β strands and α helices based on MtuDAH7PS (PDB 2B7O).

Figure S2. ITC experiments to determine ligand binding to PaeDAH7PSPA2843. A, Trp titration with PaeDAH7PSPA2843 concentration of 176 µM. B, Tyr titration with PaeDAH7PSPA2843 concentration of 100 µM. Ligands were titrated over a series of 20 injections (1 x 1.0 µL and 19 x 2.5 µL) at 298 K, with 200 rpm stirring.

Figure S3: Electron density omit maps for A, Trp (PDB 5UXM) and B, Tyr (PDB 5UXN). 2|Fo|-|Fc| is represented by the blue mesh contoured at 1 σ and |Fo|-|Fc| is represented by the green mesh contoured at 3 σ.

Figure S4. The second substantial cavity observed in the structure of PaeDAH7PSPA2843 in the absence (A) or presence (B) of the allosteric inhibitor Trp.


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Figure S5. The activity of PaeDAH7PSPA2843 Leu179Asp in the presence of up to 200 µM compared to that determined for the wild type enzyme.

Figure S6. Analysis of SEC-SAXS results for PaeDAH7PSPA2843 L179D (blue circles) and PaeDAH7PSPA2843 WT (red squares) in the absence of Trp. A, log I(q) versus q, 1σ error bars are indicated in grey. B, Guinier Plot (ln(I) versus q2). C, Kratky plot (q2⋅I(q) versus q) for the data in (A). D, P(r) versus r profiles for the data in (A). Where error bars are not visible, they are contained within the symbol.

Table S1. RMSD values obtained for the alignment between PaeDAH7PSPA2843 and MtuDAH7PS.

Table S2. SAXS parameters determined for PaeDAH7PSPA2843 L179D and PaeDAH7PSPA2843 WT in the absence of Trp.

AUTHOR INFORMATION Corresponding Author *(E.J.P) – Phone: +64 4 463 9055. E-mail: [email protected] or (G.B.J) – Phone : +64 6 356 9099 ext. 8462 Email: [email protected]


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Biochemistry

Author Contributions Designed experiments: O.W.S, E.J.P; Performed experiments: O.W.S; Analyzed data: O.W.S, G.B.J, E.J.P; Carried out AUC and analyzed AUC data: S.A.K; Wrote paper: O.W.S, G.B.J, E.J.P; Edited paper: O.W.S, G.B.J, E.J.P.

Funding Sources This study was supported by funds from the Maurice Wilkins Centre for Molecular Biodiscovery, the Biomolecular Interaction Centre and the New Zealand Marsden Fund (UoC 1105).

ACKNOWLEDGMENT Parts of this research were carried out on the MX1, MX2, and SAXS/WAXS beamlines at the Australian Synchrotron, Victoria, Australia, for which the help of the beam-line scientists is gratefully acknowledged.


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REFERENCES 1. Herrmann, K. M., and Weaver, L. M. (1999) The shikimate pathway, Annu. Rev. Plant. Biol. 50, 473-503. 2. Bentley, R. (1990) The shikimate pathway - a metabolic tree with many branches, Crit. Rev. Biochem. Mol. Biol. 25, 307-384. 3. Mentel, M., Ahuja, E. G., Mavrodi, D. V., Breinbauer, R., Thomashow, L. S., and Blankenfeldt, W. (2009) Of two make one: The biosynthesis of phenazines, ChemBioChem 10, 2295-2304. 4. Shumilin, I. A., Kretsinger, R. H., and Bauerle, R. H. (1999) Crystal structure of phenylalanine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli, Structure with Folding & Design 7, 865-875. 5. Hartmann, M., Schneider, T. R., Pfeil, A., Heinrich, G., Lipscomb, W. N., and Braus, G. H. (2003) Evolution of feedback-inhibited beta/alpha barrel isoenzymes by gene duplication and a single mutation, Proc. Natl. Acad. Sci. U. S. A. 100, 862-867. 6. Shumilin, I. A., Bauerle, R., Wu, J., Woodard, R. W., and Kretsinger, R. H. (2004) Crystal structure of the reaction complex of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation, J. Mol. Biol. 341, 455-466. 7. Schofield, L. R., Anderson, B. F., Patchett, M. L., Norris, G. E., Jameson, G. B., and Parker, E. J. (2005) Substrate ambiguity and crystal structure of Pyrococcus furiosus 3-deoxy-darabino-heptulosonate-7-phosphate synthase: An ancestral 3-deoxyald-2-ulosonatephosphate synthase?, Biochem. 44, 11950-11962.


41

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Page 42 of 48

8. Webby, C. J., Baker, H. M., Lott, J. S., Baker, E. N., and Parker, E. J. (2005) The structure of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis reveals a common catalytic scaffold and ancestry for type I and type II enzymes, J. Mol. Biol. 354, 927-939. 9. Zhou, L., Wu, J., Janakiraman, V., Shumilin, I. A., Bauerle, R., Kretsinger, R. H., and Woodard, R. W. (2012) Structure and characterization of the 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase from Aeropyrum pernix, Bioorganic Chem. 40, 7986. 10. Light, S. H., Halavaty, A. S., Minasov, G., Shuvalova, L., and Anderson, W. F. (2012) Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an n-terminal chorismate mutase-like regulatory domain, Protein Sci. 21, 887-895. 11. Chaudhury, S., Abdulhameed, M. D. M., Singh, N., Tawa, G. J., D'Haeseleer, P. M., Zemla, A. T., Navid, A., Zhou, C. E., Franklin, M. C., Cheung, J., Rudolph, M. J., Love, J., Graf, J. F., Rozak, D. A., Dankmeyer, J. L., Amemiya, K., Daefler, S., and Wallqvist, A. (2013) Rapid countermeasure discovery against Francisella tularensis based on a metabolic network reconstruction, PLoS One 8, 13. 12. Cross, P. J., Pietersma, A. L., Allison, T. M., Wilson-Coutts, S. M., Cochrane, F. C., and Parker, E. J. (2013) Neisseria meningitidis expresses a single 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase that is inhibited primarily by phenylalanine, Protein Sci. 22, 1087-1099. 13. Nazmi, A. R., Lang, E. J. M., Bai, Y., Allison, T. M., Othman, M. H., Panjikar, S., Arcus, V. L., and Parker, E. J. (2016) Interdomain conformational changes provide allosteric regulation en route to chorismate, J. Biol. Chem. 291, 21836-21847.


42

Page 43 of 48 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

Biochemistry

14. Burschowsky, D. T., H. V.; Heim, J. B.; Fahrig-Kamarauskaitė, J.; Würth-Roderer, K.; Kast, P. and Krengel U. (2018) Inter-enzyme allosteric regulation of chorismate mutase in Corynebacterium glutamicum: Structural basis of feedback activation by trp. Doi: 10.1021/acs.Biochem.7b01018. 15. Shumilin, I. A., Zhao, C., Bauerle, R., and Kretsinger, R. H. (2002) Allosteric inhibition of 3deoxy-D-arabino-heptulosonate-7-phosphate synthase alters the coordination of both substrates, J. Mol. Biol. 320, 1147-1156. 16. Aravind, L., and Koonin, E. V. (1999) Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches, J. Mol. Biol. 287, 1023-1040. 17. Webby, C. J., Jiao, W. T., Hutton, R. D., Blackmore, N. J., Baker, H. M., Baker, E. N., Jameson, G. B., and Parker, E. J. (2010) Synergistic allostery, a sophisticated regulatory network for the control of aromatic amino acid biosynthesis in Mycobacterium tuberculosis, J. Biol. Chem. 285, 30567-30576. 18. Blackmore, N. J., Reichau, S., Jiao, W. T., Hutton, R. D., Baker, E. N., Jameson, G. B., and Parker, E. J. (2013) Three sites and you are out: Ternary synergistic allostery controls aromatic amino acid biosynthesis in Mycobacterium tuberculosis, J. Mol. Biol. 425, 1582-1592. 19. Jiao, W., Blackmore, N. J., Nazmi, A. R., and Parker, E. J. (2017) Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis, PLoS One 12, 21.


43

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

Page 44 of 48

20. Sasso, S., Okvist, M., Roderer, K., Gamper, M., Codoni, G., Krengel, U., and Kast, P. (2009) Structure and function of a complex between chorismate mutase and DAHP synthase: Efficiency boost for the junior partner, Embo J. 28, 2128-2142. 21. Blackmore, N. J., Nazmi, A. R., Hutton, R. D., Webby, M. N., Baker, E. N., Jameson, G. B., and Parker, E. J. (2015) Complex formation between two biosynthetic enzymes modifies the allosteric regulatory properties of both an example of molecular symbiosis, J. Biol. Chem. 290, 18187-18198. 22. Cohen, T. S., and Prince, A. (2012) Cystic fibrosis: A mucosal immunodeficiency syndrome, Nat. Med. 18, 509-519. 23. Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S. L., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K. S., Wu, Z., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock, R. E. W., Lory, S., and Olson, M. V. (2000) Complete genome sequence of Pseudomonas aeruginosa pao1, an opportunistic pathogen, Nature 406, 959-964. 24. Perugini, M. A., Schuck, P., and Howlett, G. J. (2000) Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid, J. Biol. Chem. 275, 36758-36765. 25. Schuck, P. (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling, Biophys. J. 78, 1606-1619.


44

Page 45 of 48 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

Biochemistry

26. Schuck, P., Perugini, M. A., Gonzales, N. R., Howlett, G. J., and Schubert, D. (2002) Sizedistribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems, Biophys. J. 82, 1096-1111. 27. Kirby, N. M., Mudie, S. T., Hawley, A. M., Cookson, D. J., Mertens, H. D. T., Cowieson, N., and Samardzic-Boban, V. (2013) A low-background-intensity focusing small-angle x-ray scattering undulator beamline, J. Appl. Crystallogr. 46, 1670-1680. 28. Kirby, N., Cowieson, N., Hawley, A. M., Mudie, S. T., McGillivray, D. J., Kusel, M., Samardzic-Boban, V., and Ryan, T. M. (2016) Improved radiation dose efficiency in solution saxs using a sheath flow sample environment, Acta Crystallogr. Sect. D-Struct. Biol. 72, 1254-1266. 29. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J., and Svergun, D. I. (2003) Primus: A windows pc-based system for small-angle scattering data analysis, J. Appl. Crystallogr. 36, 1277-1282. 30. Svergun, D., Barberato, C., and Koch, M. H. J. (1995) Crysol - a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates, J. Appl. Crystallogr. 28, 768-773. 31. McPhillips, T. M., McPhillips, S. E., Chiu, H. J., Cohen, A. E., Deacon, A. M., Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P., Soltis, S. M., and Kuhn, P. (2002) Blu-ice and the distributed control system: Software for data acquisition and instrument control at macromolecular crystallography beamlines, J. Synchrotron Radiat. 9, 401-406. 32. Vagin, A., and Teplyakov, A. (1997) Molrep: An automated program for molecular replacement, J. Appl. Crystallogr. 30, 1022-1025.


45

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

Page 46 of 48

33. Emsley, P., and Cowtan, K. (2004) Coot: Model-building tools for molecular graphics, Acta Crystallogr. Sect. D-Biol. Crystallogr. 60, 2126-2132. 34. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallogr. Sect. D-Biol. Crystallogr. 53, 240-255. 35. Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state, J. Mol. Biol. 372, 774-797. 36. Kabsch, W. (1976) Solution for best rotation to relate 2 sets of vectors, Acta Crystallogr. Sect. A 32, 922-923. 37. Webby, C. J., Patchett, M. L., and Parker, E. J. (2005) Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori, Biochem. J. 390, 223-230. 38. Liu, Y. J., Li, P. P., Zhao, K. X., Wang, B. J., Jiang, C. Y., Drake, H. L., and Liu, S. J. (2008) Corynebacterium glutamicum contains 3-deoxy-D-I-heptulosonate 7-phosphate synthases that display novel biochemical features, Appl. Environ. Microbiol. 74, 5497-5503. 39. Jiao, W. T., Hutton, R. D., Cross, P. J., Jameson, G. B., and Parker, E. J. (2012) Dynamic cross-talk among remote binding sites: The molecular basis for unusual synergistic allostery, J. Mol. Biol. 415, 716-726. 40. Reichau, S., Jiao, W. T., Walker, S. R., Hutton, R. D., Baker, E. N., and Parker, E. J. (2011) Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis combining mechanism- and modeling-based design, J. Biol. Chem. 286, 11.


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41. Okvist, M., Dey, R., Sasso, S., Grahn, E., Kast, P., and Krengel, U. (2006) 1.6 a crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: Novel fold topology revealed, J. Mol. Biol. 357, 1483-1499. 42. Okvist, M., Sasso, S., Roderer, K., Kast, P., and Krengel, U. (2009) A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: Protein purification, crystallization and x-ray diffraction analysis, Acta Crystallogr. FStruct. Biol. Commun. 65, 1048-1052.


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For Table of Contents Use Only

A pseudo-isostructural type II DAH7PS enzyme from Pseudomonas aeruginosa: alternative evolutionary strategies to control shikimate pathway flux Oliver W. Sterritt, Sarah A. Kessans, Geoffrey B. Jameson and Emily J. Parker


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A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas

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