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Characterisation of the enzymes involved in the diol synthase metabolic pathway in Pseudomonas aeruginosa.

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Title: Characterisation of the enzymes involved in the diol synthase metabolic pathway in Pseudomonas aeruginosa.
Authors: Shoja-Chaghervand, Shirin1 (AUTHOR), Castells, Marc1 (AUTHOR), Rabanal, Francesc2 (AUTHOR), Cajal, Yolanda3 (AUTHOR), Manresa, Angeles1 (AUTHOR), Estupiñán, Mónica1 (AUTHOR), Busquets, Montserrat1,4 (AUTHOR) mbusquets@ub.edu
Source: Process Biochemistry. Sep2022, Vol. 120, p301-312. 12p.
Subjects: Inductively coupled plasma mass spectrometry, Pseudomonas aeruginosa, Dioxygenases, Fourier transform spectroscopy, Enzymes, Cellular inclusions
Abstract: This study is the first attempt to report the biochemical characterisation of the 10 S -dioxygenase (10 S -DOX) and 7 S , 10 S -diol synthase (7,10-DS). Both enzymes showed similar pH profiles with 10 S -DOX presenting the highest activity at 30 °C whereas 7,10-DS did not show a clear preferred temperature. These differences were reflected in the thermostability assay, the Km values were 0.89 ± 0.22 mM and 3.26 ± 0.31 mM for 10 S -DOX and 7,10-DS, respectively. Inductively coupled plasma mass spectrometry indicated that both enzymes contained bound to haem group Fe2+ as a prosthetic group: 10 S -DOX (0.95 mol Fe2+/mol of protein) and 7,10-DS (1.18 mol Fe2+/mol of protein), respectively. Assays using metal cations as cofactors revealed that Mg2+ and Ni2+ enhance 7,10-DS activity, whereas Hg2+ decrease it up to 50 %. The activity of 10 S -DOX in the presence of Mn2+ and Fe2+ was reduced to 51.6 % and 61.8. Aggregated proteins producing 10 S -DOX and 7,10-DS were characterised as inclusion bodies: IBs-77 and IBs-78 respectively, was performed by Fourier Transform spectroscopy (FT-IR), Atomic Force Microscopy, dye binding, and proteolysis. The specific activity was 1.55 IU/mg for IBs-77 and 1.05 IU/mg for IBs-78. The presence of the oleate-diol synthase pathway in proteobacteria other than Pseudomonas aeruginosa was detected. [Display omitted] • Biochemical characterisation of recombinant 10 S -dioxygenase (10 S -DOX) and 7,10-diolsynthase (7,10-DS) from P. aeruginosa. • Production of 10 S- hydroxy(per)-oxi-(8 E)-octadecenoic acid using recombinant 10 S -DOX. • Aggregate proteins 10 S -DOX and 7,10-DS were characterised as inclusion bodies. • Oleic acid conversion by diol syntase system is not restricted to P. aeruginosa. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
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Abstract:This study is the first attempt to report the biochemical characterisation of the 10 S -dioxygenase (10 S -DOX) and 7 S , 10 S -diol synthase (7,10-DS). Both enzymes showed similar pH profiles with 10 S -DOX presenting the highest activity at 30 °C whereas 7,10-DS did not show a clear preferred temperature. These differences were reflected in the thermostability assay, the Km values were 0.89 ± 0.22 mM and 3.26 ± 0.31 mM for 10 S -DOX and 7,10-DS, respectively. Inductively coupled plasma mass spectrometry indicated that both enzymes contained bound to haem group Fe2+ as a prosthetic group: 10 S -DOX (0.95 mol Fe2+/mol of protein) and 7,10-DS (1.18 mol Fe2+/mol of protein), respectively. Assays using metal cations as cofactors revealed that Mg2+ and Ni2+ enhance 7,10-DS activity, whereas Hg2+ decrease it up to 50 %. The activity of 10 S -DOX in the presence of Mn2+ and Fe2+ was reduced to 51.6 % and 61.8. Aggregated proteins producing 10 S -DOX and 7,10-DS were characterised as inclusion bodies: IBs-77 and IBs-78 respectively, was performed by Fourier Transform spectroscopy (FT-IR), Atomic Force Microscopy, dye binding, and proteolysis. The specific activity was 1.55 IU/mg for IBs-77 and 1.05 IU/mg for IBs-78. The presence of the oleate-diol synthase pathway in proteobacteria other than Pseudomonas aeruginosa was detected. [Display omitted] • Biochemical characterisation of recombinant 10 S -dioxygenase (10 S -DOX) and 7,10-diolsynthase (7,10-DS) from P. aeruginosa. • Production of 10 S- hydroxy(per)-oxi-(8 E)-octadecenoic acid using recombinant 10 S -DOX. • Aggregate proteins 10 S -DOX and 7,10-DS were characterised as inclusion bodies. • Oleic acid conversion by diol syntase system is not restricted to P. aeruginosa. [ABSTRACT FROM AUTHOR]
ISSN:13595113
DOI:10.1016/j.procbio.2022.06.017