skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structure of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas Aeruginosa

; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
Report Number(s):
Journal ID: ISSN 0006-2960
DOE Contract Number:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 54; Journal Issue: 3
Country of Publication:
United States

Citation Formats

Fedorov, A, Marti-Arbona, R, Newmmara, V, Hitchock, D, Fedorov, E, Almo, S, and Raushel, F. Structure of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas Aeruginosa. United States: N. p., 2015. Web. doi:10.1021/bi501299y.
Fedorov, A, Marti-Arbona, R, Newmmara, V, Hitchock, D, Fedorov, E, Almo, S, & Raushel, F. Structure of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas Aeruginosa. United States. doi:10.1021/bi501299y.
Fedorov, A, Marti-Arbona, R, Newmmara, V, Hitchock, D, Fedorov, E, Almo, S, and Raushel, F. 2015. "Structure of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas Aeruginosa". United States. doi:10.1021/bi501299y.
title = {Structure of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas Aeruginosa},
author = {Fedorov, A and Marti-Arbona, R and Newmmara, V and Hitchock, D and Fedorov, E and Almo, S and Raushel, F},
abstractNote = {},
doi = {10.1021/bi501299y},
journal = {Biochemistry},
number = 3,
volume = 54,
place = {United States},
year = 2015,
month = 1
  • 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 R-oxyanion carboxylate intermediate/transition state) and Mg{sup 2+} was determined at 1.9 {angstrom} 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+more » 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{sup {epsilon}} 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 {sup 1}H 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 (k{sub cat}) = 7500 s{sup -1} and K{sub m} = 2.2 mM) and 3-methyloxaloacetate (k{sub cat}) = 250 s{sup -1} and K{sub m} = 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.« less
  • The availability of high-intensity synchrotron facilities, technological advances in data-collection techniques and improved data-reduction and crystallographic software have ushered in a new era in high-throughput macromolecular crystallography. Here, the de novo automated crystal structure determination at 1.28 Angstroms resolution of an NAD(P)H-dependent FMN reductase flavoprotein from Pseudomonas aeruginosa PA01-derived protein Q9I4D4 using the anomalous signal from an unusually small number of S atoms is reported. Although this protein lacks the flavodoxin key fingerprint motif [(T/S)XTGXT], it has been confirmed to bind flavin mononucleotide and the binding site was identified via X-ray crystallography. This protein contains a novel flavin mononucleotide-binding sitemore » GSLRSGSYN, which has not been previously reported. Detailed statistics pertaining to sulfur phasing and other factors contributing to structure determination are discussed. Structural comparisons of the apoenzyme and the protein complexed with flavin mononucleotide show conformational changes on cofactor binding. NADPH-dependent activity has been confirmed with biochemical assays.« less
  • Tex is a highly conserved bacterial protein that likely functions in a variety of transcriptional processes. Here, we describe two crystal structures of the 86-kDa Tex protein from Pseudomonas aeruginosa at 2.3 and 2.5 Angstroms resolution, respectively. These structures reveal a relatively flat and elongated protein, with several potential nucleic acid binding motifs clustered at one end, including an S1 domain near the C-terminus that displays considerable structural flexibility. Tex binds nucleic acids, with a preference for single-stranded RNA, and the Tex S1 domain is required for this binding activity. Point mutants further demonstrate that the primary nucleic acid bindingmore » site corresponds to a surface of the S1 domain. Sequence alignment and modeling indicate that the eukaryotic Spt6 transcription factor adopts a similar core structure. Structural analysis further suggests that the RNA polymerase and nucleosome interacting regions of Spt6 flank opposite sides of the Tex-like scaffold. Therefore, the Tex structure may represent a conserved scaffold that binds single-stranded RNA to regulate transcription in both eukaryotic and prokaryotic organisms.« less
  • In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded {beta} barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel ({approx}8 Angstroms diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side--features likelymore » to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.« less