Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykA_PA and PykF_PA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykA_PA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykF_PA isozyme. We show that pykF _PA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykA_PA and PykF_PA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykF_PA revealed significant differences compared with PykA_PA. Notably, although the main allosteric regulator binding-site of PykF_PA was empty, the "ring loop" covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent "locked on" and "locked off" allosteric activation phenotypes, depending on the residue mutated. Analysis of PykF_PA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.

Keywords: Entner-Doudoroff pathway; Pseudomonas aeruginosa; bacterial metabolism; glycolysis; pykF; pyruvate kinase; x-ray crystallography.

FIGURE 1

Genetic context and expression of PykF. (A) ORFs associated with the PA1489-PA1502 cluster and their predicted function. (B) Structure of allantoin. (C) Predicted metabolic reactions catalyzed by each encoded enzyme in the gene cluster. Note that glyoxylate and urea are the breakdown products of allantoin degradation. (D) PykF is not expressed in wild-type P. aeruginosa in the presence of glucose but is expressed in the presence of allantoin. The figure shows western blots of cell extracts of the indicated P. aeruginosa derivatives (wild-type PAO1, pykA mutant, or pykF mutant, as indicated) obtained after overnight growth in M9 medium containing glucose, glucose plus allantoin, or allantoin alone. The blot in the upper panel was probed with anti-PykA antibodies and the blot in the lower panel was probed with anti-PykF antibodies. The identity of the cross-reacting bands with higher molecular mass than PykA or PykF in each panel is not known, but their presence in the pykA and pykF mutant extracts indicates that they are unrelated to these pyruvate kinases. (E) Allantoin-induced PykF expression is abolished in a pykF mutant. The figure shows a western blot of cell extracts from a pykA or a pykF mutant (as indicated) grown in the presence or absence of glucose and/or allantoin (as indicated). To confirm minimal cross-reactivity of the antibodies, purified PykA or PykF (as indicated) were loaded on the right-hand side of each blot.

FIGURE 2

Kinetic characterization of PykF. (A) SDS-PAGE gel showing purified, untagged PykF (51.5 kDa). The gel was prepared with 12% (w/v) acrylamide and stained with Coomassie Brilliant Blue G250 [The minor bands at around 30 and 40 kDa molecular mass correspond to degradation products of PykF]. (B) PykF kinetics with respect to PEP and ADP. The PEP titration was performed using 2 mM ADP (i.e., a saturating concentration) whereas the ADP titration was performed using 5 mM PEP (also a saturating concentration). The R² values for the curve fit to the PEP and ADP titrations were 0.96 in both cases. The 95% confidence interval estimates for the kinetic parameters are shown in Supplementary Table 4. (C) The effect of different metabolic regulators on PykF activity at low [PEP] (0.3 mM) and 2 mM ADP. Putative regulators were added at 1 mM final concentration, except for R5P, X5P, and RL5P which were used at 0.15, 0.5, and 0.5, respectively. The aim of this experiment was to identify potential activators. Data in panels (B,C) represent the mean and standard deviation of three independent experiments.

FIGURE 3

The metabolic regulation of PykF. (Left panel) Michaelis-Menten plots showing that the indicated regulators primarily convert PykF from sigmoidal to hyperbolic kinetics. Data represent the mean and standard deviation of three independent experiments. (Right panel) Lineweaver-Burk plots showing that the indicated regulators primarily act to decrease S₀.₅ of PykF compared with the control. “Control” indicates the reaction kinetics in the absence of added regulators. The changes in PykF kinetic constants elicited by each regulator are shown in Table 1. The 95% confidence interval estimates for the kinetic parameters are provided in Supplementary Table 4.

FIGURE 4

X-ray crystal structure of PykF. (Top diagrams) Front and side views of the PykF homotetramer. Chain A and B were already present in the asymmetric unit of PykF, whereas chains C and D were generated using symmetry coordinates. The A-A and the C-C interfaces are shown. The Cα1′ helices across the A-A interface are shown in dashed ovals. (Bottom diagram) Domain organization of a PykF subunit. A-, B- and C-domains of PykF are colored in blue, pink, and green, respectively. The active site helix and Cα1′ are colored in red and orange, respectively.

FIGURE 5

The active and allosteric sites of PykF. (A) (i) Cartoon representation of the crystal structure of a presumed inactive conformation of pyruvate kinase (PykF_PA; 7OO1, blue) overlain onto the crystal structure of a presumed active configuration of the enzyme (PykA_PA; 6QXL, cyan). (ii) Cartoon representation of the crystal structure of PykF_PA (inactive configuration, 7OO1, blue) overlain onto the crystal structure of PykF_EC (inactive configuration, 1PKY, yellow). (B) Surface representation of the crystal structure of PykA_PA [(i) 6QXL] and PykF_PA [(ii) 7OO1, blue]. (C) (i) Cross-section through the active site of PykA_PA (active configuration of the enzyme, 6QXL) The substrate analog, malonate, is shown in yellow. (ii) Cross-section through the active site of PykF_PA (inactive configuration of the enzyme, 7OO1). The superimposed binding mode of the malonate from 6QXL is shown in yellow. (D) Partial closure of the allosteric site of PykF_PA. Superposition of the allosteric site in PykF_PA (7OO1, blue), PykF_EC (1PKY, yellow), and PykA_PA (6QXL, cyan) showing disposition of the ring loop of PykF_PA toward the allosteric site, probably determined by the configuration of Pro455 and Pro459. The G6P bound to PykA_PA is shown as pink sticks.

FIGURE 6

The A-A interface of PykF. (A) Secondary structures present at the A-A interface in PykF. The Cα1′ helices are shown in coral. (B) The interlocking of α-helices Aα6 and Aα7 at the A-A interface (upper and lower panels show a cross-section through the interface at different tilt angles). (C) Close-up view of the interactions at the Cα1′-Cα1′ interspace in bacterial species that have a Cα1′-like structure. (D) Close-up view of the A-A interface in PykF showing salt bridge formation across the interface. Of note, the A-A interface of E. coli PykF does not contain a Cα1′-like structure or salt bridges.

FIGURE 7

Secondary structures of the C-C interface of PykF_PA (blue), PykF_EC (wheat) and PykA_PA (cyan). The diagram shows that the C-C interface in PykF_PA adopts almost the same configuration as the C-C interface in PykF_EC, likely due to the absence of bound regulator in the allosteric site. The G6P bound to PykA_PA is shown as pink sticks.

FIGURE 8

The inter-protomer interfaces in PykF. The top diagram shows a comparison between the A-A interface in PykF_PA, and (left) PykF_EC and (right) Pyk_Mtb. The bottom panel shows a comparison between the C-C interface in PykF_PA and (left) PykF_EC and (right) Pyk_Mtb. Interactions unique to PykF_PA (black dashed lines), PykF_EC (yellow dashed lines), and Pyk_Mtb (pink dashed lines) are shown. Interactions present in PykF_PA and any of the two structures are shown as solid black lines.