Structure and function of PA4872 from Pseudomonas aeruginosa a novel class of oxaloacetate decarboxylase from the PEP mutase / isocitrate lyase superfamily

摘要

Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analog of the shared family α-oxyanion carboxylate intermediate/transition state) and Mg²⁺ was determined at 1.9 Å resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an α/β barrel fold and two subunits swapping their barrel's C-terminal α-helices. Mg²⁺ and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The N^ε of His235, an invariant residue in the PA4872 sequence family, is oriented towards a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into α-oxocarboxylate-containing compounds was confirmed by ¹H-NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an α-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (kcat = 7500 s⁻¹ and Km = 2.2 mM) and 3-methyloxaloacetate (kcat = 250 s⁻¹ and Km = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gram-negative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.