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Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

ABSTRACT: The metabolism of arginine towards ATP synthesis has been considered a major source of energy for microorganisms such as Mycoplasma penetrans in anaerobic conditions. Additionally, this pathway has also been implicated in pathogenic and virulence mechanism of certain microorganisms, i.e. protection from acidic stress during infection. In this work we present the crystal structures of the three enzymes composing the gene cluster of the arginine deiminase pathway from M. penetrans: arginine deiminase (ADI), ornithine carbamoyltransferase (OTC) and carbamate kinase (CK). The arginine deiminase (ADI) structure has been refined to 2.3 Å resolution in its apo-form, displaying an "open" conformation of the active site of the enzyme in comparison to previous complex structures with substrate intermediates. The active site pocket of ADI is empty, with some of the catalytic and binding residues far from their active positions, suggesting major conformational changes upon substrate binding. Ornithine carbamoyltransferase (OTC) has been refined in two crystal forms at 2.5 Å and 2.6 Å resolution, respectively, both displaying an identical dodecameric structure with a 23-point symmetry. The dodecameric structure of OTC represents the highest level of organization in this protein family and in M.penetrans it is constituted by a novel interface between the four catalytic homotrimers. Carbamate kinase (CK) has been refined to 2.5 Å resolution and its structure is characterized by the presence of two ion sulfates in the active site, one in the carbamoyl phosphate binding site and the other in the ?-phosphate ADP binding pocket of the enzyme. The CK structure also shows variations in some of the elements that regulate the catalytic activity of the enzyme. The relatively low number of metabolic pathways and the relevance in human pathogenesis of Mycoplasma penetrans places the arginine deiminase pathway enzymes as potential targets to design specific inhibitors against this human parasite.

SUBMITTER: Gallego P

PROVIDER: S-EPMC3474736 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

Gallego Pablo P Planell Raquel R Benach Jordi J Querol Enrique E Perez-Pons Josep A JA Reverter David D

PloS one 20121017 10

The metabolism of arginine towards ATP synthesis has been considered a major source of energy for microorganisms such as Mycoplasma penetrans in anaerobic conditions. Additionally, this pathway has also been implicated in pathogenic and virulence mechanism of certain microorganisms, i.e. protection from acidic stress during infection. In this work we present the crystal structures of the three enzymes composing the gene cluster of the arginine deiminase pathway from M. penetrans: arginine deimin ...[more]

PMID: 23082227

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Project description:The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.

Dataset Information

Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

Publications

Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

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