An allantoin-inducible glyoxylate utilization pathway in Pseudomonas aeruginosa

Abstract

Fluorescent pseudomonads catabolize purines via uric acid and allantoin, a pathway whose end-product is glyoxylate. In this work, we show that in Pseudomonas aeruginosa strain PAO1, the ORFs PA1498-PA1502 encode a pathway that converts the resulting glyoxylate into pyruvate. The expression of this cluster of ORFs was stimulated in the presence of allantoin, and mutants containing transposon insertions in the cluster were unable to grow on allantoin as a sole carbon source. The likely operonic structure of the cluster is elucidated. We also show that the purified proteins encoded by PA1502 and PA1500 have glyoxylate carboligase (Gcl) and tartronate semialdehyde (TSA) reductase (GlxR) activity, respectively, in vitro. Gcl condenses two molecules of glyoxylate to yield TSA, which is then reduced by GlxR to yield d-glycerate. GlxR displayed much greater specificity (k _cat/K_M) for Gcl-derived TSA than it did for the TSA tautomer, hydroxypyruvate. This is relevant because TSA can potentially spontaneously tautomerize to yield hydroxypyruvate at neutral pH. However, kinetic and [¹H]-NMR evidence indicate that PA1501 (which encodes a putative hydroxypyruvate isomerase, Hyi) increases the rate of the Gcl-catalysed reaction, possibly by minimizing the impact of this unwanted tautomerization. Finally, we use X-ray crystallography to show that apo-GlxR is a configurationally flexible enzyme that can adopt two distinct tetrameric assemblies in vitro.

Keywords: Pseudomonas aeruginosa; allantoin; glyoxylate; metabolism; purine catabolism.

Fig. 1.. Allantoin metabolism in P. aeruginosa. (a) Putative pathway of purine degradation to glyoxylate in P. aeruginosa. Note that not all of the enzymes in this pathway have been biochemically characterized and are proposed based on amino acid similarity with those from other organisms. (b) Genetic context of the ORFs involved in purine degradation in P. aeruginosa. Blue, ORFs involved in the conversion of glyoxylate to 2-phosphoglycerate (investigated in the current work); green, ORFs predicted to encode enzymes involved in the purine/allantoin degradation pathway shown in (a); red, regulatory proteins; orange, ORFs encoding enzymes involved in glycolate oxidation; white, other. The two rows of ORFs represent one contiguous sequence. Diagram not to scale. (c) Proposed pathway of glyoxylate metabolism catalysed by the gcl cluster ORFs [shown in blue here and in (b)].

Fig. 2.. Disruption of ORFs in the gcl cluster impairs growth on allantoin as a sole carbon source. The figure shows growth (monitored as OD at 600 nm) of the progenitor strain (PA01) (black dots) and glxR::Tn (orange dots), hyi::Tn (blue/cyan dots) and gcl::Tn (pink/magenta dots) mutants in M9 minimal media supplemented with (a) 0.25% (w/v) glucose or (b) 0.33% (w/v) allantoin. The data represent the mean and sd of three biological replicates.

Fig. 3.. Expression from the gcl promoter is induced by allantoin. β-Galactosidase activity in cultures of PAO1 containing chromosomally integrated P_gcl-lacZ (or, as a control, chromosomally integrated empty miniCTX-lacZ vector; P₀-lacZ) harvested at (a) mid-log phase growth or (b) stationary phase. The growth medium was M9 medium supplemented with 0.33% (w/v) allantoin (green bars) or 0.25% (w/v) glucose (pink bars). β-Galactosidase activity is expressed in OD₄₂₀ min⁻¹ ml⁻¹ OD₆₀₀⁻¹ (Miller units), and the data represent the mean and sd of three biological replicates. Significance was calculated using unpaired t-tests and is indicated by **** (P≤0.0001) and *** (P≤0.001). (c) Computationally predicted operon structure of the gcl cluster.

Fig. 4.. Reactions catalysed by GlxR and Hyi. Gcl catalyses the dimerization of two molecules of glyoxylate to form TSA. This TSA has two possible fates. (a) Hyi catalyses the conversion of TSA into hydroxypyruvate, which is then converted by GlxR into d-glycerate. (b) GlxR reduces TSA directly to yield d-glycerate. However, in a competing reaction, TSA also spontaneously tautomerizes to yield hydroxypyruvate. In this scenario, Hyi catalyses the conversion of hydroxypyruvate to TSA, thereby restoring TSA levels and increasing the rate of the GlxR-catalysed reaction. (c) GlxR-catalysed conversion of hydroxypyruvate into d-glycerate. The concentration of GlxR was 0.4 µg ml⁻¹. Inset: Impact of Hyi addition on GlxR-catalysed reaction rate in the presence of 20 mM hydroxypyruvate. (d) The GlxR-catalysed conversion of de novo synthesized TSA from the Gcl-catalysed reaction into d-glycerate is increased in the presence of Hyi. Gcl was present at 20 μg ml⁻¹ and GlxR at 0.4 μg ml⁻¹.

Fig. 5.. The figure shows [¹H]-NMR spectra for the indicated compounds in the presence of the indicated enzymes, incubated for the indicated periods of time at 37 °C in D₂O. (a) Spectrum of hydroxypyruvate on its own. (b) Spectrum of hydroxypyruvate in the presence of Hyi, which resulted in an increased rate of disappearance of the hydroxypyruvate signals (3.65 and 4.69 p.p.m.), likely due to continuous isomerization and exchange of hydrogens for deuterium atoms. (c) Control NMR of an aliquot of Hyi (in the absence of hydroxypyruvate) in D₂O, indicating that the peak at 3.71 p.p.m. is derived from the enzyme preparation. (d) Spectrum of glyoxylate on its own. (e) Spectrum of glyoxylate in the presence of Gcl. Note the slow disappearance of the glyoxylate peak at 5.06 p.p.m. (f) Spectrum of glyoxylate in the presence of Gcl and Hyi, which resulted in rapid disappearance of the glyoxylate signal. (g) Control NMR of an aliquot of Gcl in D₂O, again indicating that the peak at 3.71 p.p.m. is derived from the enzyme preparation.

Fig. 6.. Structures of GlxR from P. aeruginosa. (a) X-ray crystal structures of GlxR α (left panel) and β (right panel) forms. The protomers in each structure are represented in different colours, and the tetramers are rotated through 180°, as indicated. (b) Protomers (left panel) and tetramers (right panel) of the α (grey) and β (brown) forms of GlxR superimposed. The root mean square deviation (RMSD) for superposition of the promoters is 2.24 Å and for superposition of the tetramers is 3.60 Å. (c) Protomers (left panel) and tetramers (right panel) of the α form of GlxR (grey) superimposed on the structure of GarR_Sty (1VPD, green). The RMSD for superposition of the protomers is 3.02 Å and for superposition of the tetramers is 5.82 Å. (d) Protomers (left panel) and tetramers (right panel) of the β form of GlxR (brown) superimposed on the structure of GarR_Sty (1VPD, green). The RMSD for superposition of the protomers is 1.68 Å and for superposition of the tetramers is 3.34 Å. (e) Protomer of the GlxR α conformer (upper panel, grey) and the GlxR β conformer (lower panel, brown) superimposed on the structure of GlxR predicted by AlphaFold (AF-Q9I3L2, blue). The RMSD for superposition of the α conformer is 1.30 Å and for superposition of the β conformer is 1.57 Å.

Fig. 7.. Sequence alignment of GlxR from P. aeruginosa and GarR from S. enterica Typhimurium. Secondary structural elements derived from the X-ray crystal structure of GlxR are indicated, as are the conserved active site residues (boxed in green).