Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Dedicated pathway for the degradation of (+)- and (-)-camphor (1) by Pseudomonas putida ATCC 17453. Key idiosyncratic pathway intermediates shown are the enantiomeric diketocamphanes (2, DKCs), the monocyclic ketone 2-oxo-△ 3,4 -4,5,5-trimethylcyclopentenylacetyl-CoA (3, HTP-CoA) itself formed from the corresponding transitory free acid precursor OTE (2-oxo-△ 3,4 -4,5,5-trimethylcyclopentenylacetic acid), and the unstable monocyclic lactone 5-hydroxy-3,4,4-trimethyl-△ 2 -pimelyl- δ -CoA lactone (4). Subsequent hydrolysis of the monocyclic lactone generates the first aliphatic intermediate, a C10 carboxylic acid that is further metabolised to the TCA cycle intermediates acetyl-CoA and succinyl-CoA. The key sequential enzyme-catalysed steps are: a. camphor 5-monooxygenase ( camCAB ); b. exo hydroxycamphor dehydrogenase ( camD ); c. 2,5-diketocamphane 1,2-monooxygenase ( camE 25-1 + camE 25-2 ); d. 3,6-diketocamphane 1,6-monooxygenase ( camE 36 ); e. 2-oxo-△ 3,4 -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase ( camG ).

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques:

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Localization of additional genes and predicted open reading frames (ORFs) flanking the established initial genes of the camphor camDCAB operon and its repressor, camR , on an~40.5-kb established sequenced region of the CAM plasmid of P. putida ATCC 17453. The predicted ORFs or genes are sequenced numbered from 1 to 27, except for the established camRDCAB genes, which are shaded in black. The numbered from 1 to 27, except for the established camRDCAB genes, which are shaded in black. The orientation of the arrows indicates the direction of gene transcription. The candidate genes of this study ( camE 25–1 , camE 25–2 , and camE 36 ) representing the three diketocamphane monooxygenase (DKCMO) isozymes are highlighted in grey. The previously established OTEMO-encoding gene been designated camG in accordance with the respective catabolic step. Figure reproduced from Iwaki et al. with permission. American Society for Microbiology, licence number 4487631509592.

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques: Plasmid Preparation

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Changes in the optical density (A 500 nm), succinate (mM), ( rac )-camphor (mM), and the specific activity of key enzymes of ( rac )-camphor degradation during diauxic growth of by P. putida ATCC 17453 on succinate plus ( rac )-camphor based defined medium. Camphor hydroxylase = camphor 5-monooxygenase.

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques: Activity Assay

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Transcriptional controls of the pathway of (+)- and (−)-camphor degradation in P. putida ATCC 17453 ●+ = induction: −● = repression: A = camphor 5-monooxygenase ( camCAB ): B = exo -hydroxycamphor dehydrogenase ( camD ): C = 2,5-diketocamphane 1,2-monooxygenase ( camE 25-1 + camE 25-2 ); D = 3,6-diketo- camphane 1,6-monooxygenase camE 36 ); E = 2-oxo-△ 3 -4,5,5-trimethylcyclo-pentenylacetyl-CoA synthetase ( camF1 + F2) ; F = 2-oxo-△ 3 -4,5,5-trimethylcyclo-pentenylacetyl-CoA monooxygenase ( camG ).

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques:

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: The relationships between nonselective and stereoselective enzymes that constitute the pathway for the degradation of ( rac )-camphor to OTE in P. putida ATCC 17453, and the roles of chiral and achiral molecules as substrates and transcriptional regulators. A = induction by (+)-camphor and (−)-camphor, plus product induction by OTE: B = induction by (+)-camphor and (−)-camphor, plus product induction by 2,5-DKC and 3,6-DKC: C = induction by (+)-camphor, (−)-camphor and 2,5-DKC, plus cross-induction by 3,6-DKC, and product induction by OTE: D = ‘from the top’ coordinate induction by (+)-camphor, (−)-camphor, and 3,6-DKC, plus cross-induction by 2,5-DKC, and product induction by OTE: A + B + C + D = ‘from the top’ coordinate induction by (+)-camphor and (−)-camphor.

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques:

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Key interrelationships between CAM plasmid-coded functions and chromosome-coded functions in ( rac )-camphor-grown P. putida ATCC 17453. black = biomolecules exclusive to CAM plasmid-directed biochemistry: red = biomolecules exclusive to chromosome-directed biochemistry: green = biomolecules common to both plasmid-directed and chromosome-directed biochemistry. DKC = diketocamphane; FNR = FMNH 2. ; cytP540MO = camphor 5-monooxygenase; OTE = 2-oxo-△ 3 -4,5,5-trimethylcyclopentylacetic acid; PdR = putidaredoxin.

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques: Plasmid Preparation

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Changes in the activity of ( A ) camphor 5-monooxygenase (cytP450MO), and ( B ) the combined Frp1 + Frp2 activity in P. putida ATCC 17453 in the presence and absence of 30 µM Zn 2+ added during the diauxic interlude that occurs between the swop from late log growth on succinate to early log growth on ( rac )-camphor.

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques: Activity Assay

Journal: Microorganisms

Article Title: The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

doi: 10.3390/microorganisms9122593

Figure Lengend Snippet: Schematic of the two contrasting roles of putidaredoxin reductase (PdR) in camphor-grown P. putida ATCC 17453. ( A ). cytP450MO (camphor 5-monooxygenase): Pdx (putidaredoxin) ( B ). 2,5-DKCMO (2,5-diketocamphane 1,2-monooxygenase): DKC (2,5-diketocamphane).

Article Snippet: Notably, the presence of Zn 2+ resulted in no significant difference to either biomass yield, or the activity profiles of the 2,5- and 3,6-DKCMOs, and as expected for trophophasic phase cultures of ( rac )-camphor-grown P. putida ATCC 17453, no detectible titres of Fred were recorded in any of the tested biomass samples [ ].

Techniques:

Journal: Biochimica et biophysica acta. General subjects

Article Title: Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450 cam (CYP101A1).

doi: 10.1016/j.bbagen.2018.10.018

Figure Lengend Snippet: Fig. 1. a) Reported steps involved in the catabolic degradation of camphor by Pseudomonas putida (ATCC 17453). a Chemical structures of (+)-borneol, (L)-glutamic acid, (D)-glutamic acid, and 1-phenylimidazole. 1= (+)-camphor; 2 = 5-exo-hydroxycamphor; 3 = 5-ketocamphor; 4 = unstable lactone; 5 = 2-oxo-Δ3–4,5,5-trimethylcyclopentenylacetic acid; 6 = 2-oxo- Δ3–4,5,5-tri-methylcyclopentenylacetyl-CoA; 7 = 3,4,4-trimethyl-Δ3-pimelyl-CoA; 8 = (+)-borneol; 9 = (L)-glutamicacid; 10 = (D)-glutamic acid; 11 = 1-phe- nylimidazole; A = P450cam, PdR, & PdX; B = 5-exo-hydroxycamphor dehydrogenase; C = 2,5-diketocamphane monooxygenase; D = 2-oxo-Δ3–4,5,5-tri- methylcyclo-pentenylacetyl-CoA synthetase; E = 2-oxo-Δ3–4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase.

Article Snippet: In-plug chemotaxis assays of P. putida strains (ATCC 17453, 17,484, & 33,015) The chemotactic responses of three wild, aerobic,P. putidasoil Strain 2= Pseudomonas putida ATCC 17484 (naphthalene-metabolizing).

Techniques:

Journal: Biochimica et biophysica acta. General subjects

Article Title: Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450 cam (CYP101A1).

doi: 10.1016/j.bbagen.2018.10.018

Figure Lengend Snippet: Fig. 2. Effects of1-phenylimidazole (a P450cam inhibitor) on growth P. putida ATCC 17453 (Strain 1). A)Survival dose response of Strain 1treated with 1-phenyli- midazole. B)Growthof Strain 1 on minimal medium with1-phenylimidazole as a sole carbon source. Each point represents the mean ± S. E. of 3 replicates.

Article Snippet: In-plug chemotaxis assays of P. putida strains (ATCC 17453, 17,484, & 33,015) The chemotactic responses of three wild, aerobic,P. putidasoil Strain 2= Pseudomonas putida ATCC 17484 (naphthalene-metabolizing).

Techniques:

Journal: Biochimica et biophysica acta. General subjects

Article Title: Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450 cam (CYP101A1).

doi: 10.1016/j.bbagen.2018.10.018

Figure Lengend Snippet: Fig. 4. Survival assay with (+)-camphor in the presence and absence of 1-phenylimidazole (a cytochrome P450cam inhibitor).A) Dose response for P. putida ATCC 17453 treated with (+)-camphor. B) Dose response for P. putida ATCC 17453, grown in the presence of 1-phenylimidazole (20 mM) and treated with (+)-camphor. Each point represents the mean ± S. E. of 3 replicates.

Article Snippet: In-plug chemotaxis assays of P. putida strains (ATCC 17453, 17,484, & 33,015) The chemotactic responses of three wild, aerobic,P. putidasoil Strain 2= Pseudomonas putida ATCC 17484 (naphthalene-metabolizing).

Techniques: Clonogenic Cell Survival Assay

Journal: EMBO Molecular Medicine

Article Title: The clinical heterogeneity of coenzyme Q 10 deficiency results from genotypic differences in the Coq9 gene

doi: 10.15252/emmm.201404632

Figure Lengend Snippet: CoQ biosynthetic gene expression A–E mRNA expression levels of Coq9 (A), Coq7 (B), Adck3 (C), Coq5 (D) and Coq6 (E) on cerebrum of Coq9 +/+ , Coq9 Q95X and Coq9 Q95X mice at 3 months of age. ** P < 0.01; *** P < 0.001; Coq9 Q95X and Coq9 R239X mice versus Coq9 +/+ mice. ### P < 0.001; Coq9 Q95X versus Coq9 R239X mice. F–J mRNA expression levels of Coq9 (F), Coq7 (G), Adck3 (H), Coq5 (I) and Coq6 (J) on kidney of Coq9 +/+ , Coq9 Q95X and Coq9 Q95X mice at 3 months of age. *** P < 0.001; Coq9 Q95X and Coq9 R239X mice versus Coq9 +/+ mice. # P < 0.05; ### P < 0.001; Coq9 Q95X versus Coq9 R239X mice. K–O mRNA expression levels of Coq9 (K), Coq7 (L), Adck3 (M), Coq5 (N ) and Coq6 (O) on triceps surae of Coq9 +/+ , Coq9 Q95X and Coq9 Q95X mice at 3 months of age. * P < 0.05; *** P < 0.001; Coq9 Q95X and Coq9 R239X mice versus Coq9 +/+ mice. # P < 0.05; Coq9 Q95X versus Coq9 R239X mice. Data information: All values are presented as mean ± SD. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P -values of the one-way ANOVA test ( n = 5 for each group).

Article Snippet: The following primary antibodies were used: anti-COQ7 (generously provided by Dr Hekimi, McGill University, Canada), anti-COQ6 (Santa Cruz Biotechnology, sc-393932), anti-COQ5 (Proteintech™, 17453-1-AP), anti-ADCK3 (Abnova, M04A) anti-COQ9 (Santa Cruz, sc-271892), anti-COQ9 (Abcam, ab104189) and anti-VDAC1 (Abcam, ab14734).

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: The clinical heterogeneity of coenzyme Q 10 deficiency results from genotypic differences in the Coq9 gene

doi: 10.15252/emmm.201404632

Figure Lengend Snippet: Levels of COQ biosynthetic proteins A–D Representative Western blot and quantitation of Western blot bands of COQ7 (A), ADCK3 (B), COQ5 (C) and COQ6 (D), and VDAC1 as a loading control in the kidneys of 3-month-old mice. * P < 0.05; ** P < 0.01; *** P < 0.001; Coq9 Q95X and Coq9 R239X mice versus Coq9 +/+ mice. ## P < 0.01; ### P < 0.001; Coq9 Q95X versus Coq9 R239X mice. One-way ANOVA with a Tukey's post hoc test. E–H Representative Western blot and quantitation of Western blot bands of COQ7 (E), ADCK3 (F), COQ5 (G) and COQ6 (H), and VDAC1 as a loading control in skeletal muscle of 3-month-old mice. ** P < 0.01; *** P < 0.001; Coq9 Q95X and Coq9 R239X mice versus Coq9 +/+ mice. # P < 0.05; ## P < 0.01; Coq9 Q95X versus Coq9 R239X mice. Data information: All values are presented as mean ± SD. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P -values of the one-way ANOVA test. Coq9 +/+ mice n = 4; Coq9 Q95X and Coq9 R239X mice n = 5. Source data are available online for this figure.

Article Snippet: The following primary antibodies were used: anti-COQ7 (generously provided by Dr Hekimi, McGill University, Canada), anti-COQ6 (Santa Cruz Biotechnology, sc-393932), anti-COQ5 (Proteintech™, 17453-1-AP), anti-ADCK3 (Abnova, M04A) anti-COQ9 (Santa Cruz, sc-271892), anti-COQ9 (Abcam, ab104189) and anti-VDAC1 (Abcam, ab14734).

Techniques: Western Blot, Quantitation Assay