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

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TitleStructure and function of PA4872 from Pseudomonas aeruginosa, a novel class of oxaloacetate decarboxylase from the PEP mutase/isocitrate lyase superfamily.
Publication TypeJournal Article
Year of Publication2008
AuthorsNarayanan, BC, Niu, W, Han, Y, Zou, J, Mariano, PS, Dunaway-Mariano, D, Herzberg, O
JournalBiochemistry
Volume47
Issue1
Pagination167-82
Date Published2008 Jan 8
ISSN0006-2960
KeywordsAmino Acid Sequence, Bacterial Proteins, Bicarbonates, Binding Sites, Carboxy-Lyases, Crystallography, X-Ray, Dimerization, Isocitrate Lyase, Magnesium, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Molecular Structure, Mutagenesis, Site-Directed, Oxalates, Oxaloacetic Acid, Phosphotransferases (Phosphomutases), Protein Structure, Secondary, Protein Structure, Tertiary, Pseudomonas aeruginosa, Pyruvates, Sequence Homology, Amino Acid, Structure-Activity Relationship, Substrate Specificity
Abstract

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 analogue of the shared family alpha-oxyanion carboxylate intermediate/transition state) and Mg2+ was determined at 1.9 A resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an alpha/beta barrel fold and two subunits swapping their barrel's C-terminal alpha-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 Nepsilon of His235, an invariant residue in the PA4872 sequence family, is oriented toward a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into alpha-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 alpha-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.

DOI10.1021/bi701954p
Alternate JournalBiochemistry
PubMed ID18081320
PubMed Central IDPMC2892964
Grant ListR01 GM028688-29 / GM / NIGMS NIH HHS / United States
R01GM28688 / GM / NIGMS NIH HHS / United States