Investigation into the Mode of Phosphate Activation in the 4-Hydroxy-4-Methyl-2-Oxoglutarate/4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase from Pseudomonas putida F1

Abstract

The 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is the last enzyme of both the gallate and protocatechuate 4,5-cleavage pathways which links aromatic catabolism to central cellular metabolism. The enzyme is a class II, divalent metal dependent, aldolase which is activated in the presence of inorganic phosphate (Pi), increasing its turnover rate >10-fold. This phosphate activation is unique for a class II aldolase. The aldolase pyruvate methyl proton exchange rate, a probe of the general acid half reaction, was increased 300-fold in the presence of 1 mM Pi and the rate enhancement followed saturation kinetics giving rise to a KM of 397 ± 30 μM. Docking studies revealed a potential Pi binding site close to, or overlapping with, the proposed general acid water site. Putative Pi binding residues were substituted by site-directed mutagenesis which resulted in reductions of Pi activation. Significantly, the active site residue Arg-123, known to be critical for the catalytic mechanism of the enzyme, was also implicated in supporting Pi mediated activation.

CH‐π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

“…Analysis over a broad range of concentrations revealed a considerable affinity towards phosphate, with an apparent dissociation constant of K d,Pi =175±36 μM. This observation was of particular interest, since phosphate buffers were reported to exhibit an adverse effect on the activity of class II aldolases in several other cases, with only one exception . Strikingly, even minor concentrations of phosphate significantly increased the reaction rate up to 13‐fold under standard assay conditions, from 2.1 U/mg to v sat =27.1 U/mg following saturation kinetics with respect to phosphate (Figure ).…”

“…In theory, structural analogues of phosphate with matching pKa values should also be able to act as the catalytic base. Indeed, AsO 4 3− and VO 4 3− were previously shown to similarly activate the HMG/CHA aldolase, while SO 4 2− and MoO 4 2− did not . A brief overview of phosphate analogues and their corresponding pKa values is given in Table S3.…”

“…While a recent study on the class II HMG/CHA pyruvate aldolase described a 10‐fold rate enhancement by inorganic phosphate, these findings were attributed to the enzyme's distinct structure (αββα sandwich fold in the HMG/CHA aldolase vs . an (α/β) 8 barrel fold in the DDG aldolase; 16.8% shared sequence identity) . Here we present new insights into the mode of phosphate activation by identifying the key residues that are involved in the binding of phosphate and the concomitant rate enhancement.…”

Substrate Induced Movement of the Metal Cofactor between Active and Resting State

“…The activity assay is based on the retro‐aldol type decarboxylation of oxaloacetate and displayed a significantly lower rate acceleration by phosphate (approx. 10 fold for both Sw HKA and HMG/CHA aldolase),[ 15 , 23 ] and examines a different catalytic step (Figure 3 b). Indeed, the oxaloacetate must bind partly in the position occupied by the phosphate in the docking.…”

Substrate Induced Movement of the Metal Cofactor between Active and Resting State

“…The activity assay is based on the retro-aldol type decarboxylation of oxaloacetate and displayed a significantly lower rate acceleration by phosphate (approx. 10 fold for both SwHKA and HMG/ CHA aldolase), [15,23] and examines a different catalytic step (Figure 3b). Indeed, the oxaloacetate must bind partly in the position occupied by the phosphate in the docking.…”