Toluene: Oxidation and ring opening

1

• Toluene Oxidation and ring opening
• Naphthalene

2
Toluene Toluene, xylene, and similar aromatic
compounds are degraded by enzymes of the TOL
plasmids, for example pWW0, the best studied
metabolic plasmid (SJ Assinder, Adv. Micr.
Biol., 1990).
3

• Toluene Toluene, xylene, and similar aromatic
  compounds are degraded by enzymes of the TOL
  plasmids, for example pWW0, the best studied
  metabolic plasmid (SJ Assinder, Adv. Micr.
  Biol., 1990).

4

• Toluene
• Substrates are oxidized on the side chain (upper
  pathway), hydroxylated to catechols and cleaved
  (meta pathway).

upper pathway meta
pathway regulatory
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• Toluene
• Upper pathway stepwise oxidation to benzoates.
  Notice the requirement for NADH in the first
  step.

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Toluene Benzoate can be metabolized via
b-ketoadipate, but this is not possible for
derivatives of benzoate gt an additional pathway
is needed.
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• Toluene
• Meta pathway

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• Toluene Catechol-2,3-dioxygenase, XylE, C23O,
  Metapyrocatechase

Tetramer, 4 x 35 kDa Oxygen sensitive, but
protected by EtOH or acetone One Fe2/monomer
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• Toluene Catechol-2,3-dioxygenase, Fe coordination

Fe is coordinated by His, His, Glu. Acetone is
bound to the active site.
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• Toluene substrate binding site of
  catechol-2,3-dioxygenase

The pocket for the substrate is hydrophobic Phe,
Leu, Ile, Thr, Ala
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• Toluene anaerobic degradation
• Problem Activation of the methyl group - Easier
  with O2, more difficult without!
• gtgt benzylsuccinate synthase

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• Toluene mechanism of action of benzylsuccinate
  synthase

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• Toluene benzyl succinate synthase
• The gene cluster contains four genes bssA-D.
• BssABC encode the benzyl succinate synthase, BssD
  the activating enzyme (in Thauera aromatica).

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• Toluene benzyl succinate synthase
• The subunits a, b, and g are encoded by BssA-C.
• BssA is similar to pyruvate formate lyase (PflD)
  and contains a glycyl radical.

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• Toluene benzyl succinate synthase
• The radical stems from Gly825 of subunit a , 25
  aa from the C-terminus, a conserved residue in
  glycyl radical enzymes. The enzyme is very O2
  sensitive (t1/2 30s). The main chain can be
  cleaved at this position, producing the slightly
  smaller a peptide.

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• Aromatic ring cleavage under anaerobic
  conditions
• Further metabolism of benzoyl-CoA proceeds via
  reduction of the aromatic ring, b-oxidation
  steps, ring cleavage, and subsequent
  mineralization via 3-oxopimelyl-CoA and
  glutaryl-CoA.

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Naphthalene Direct dioxygenation of the
ring Both oxygen atoms are added
stereospecifically to give exclusively cis diols.

The Pseudomonas enzyme only produces the (1R,2S)
diol (left).
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Naphthalene Direct dioxygenation of the ring An
Fe2 is coordinated by His, His, Asp. Oxygen
binds side-on to the iron, in place for attack.
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Naphthalene Direct dioxygenation of the ring The
bound product, naphthalene cis-dihydrodiol
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Naphthalene The Rieske center (2Fe-2S) and the
Fe2 reduce O2 to O22-. O-O distance in the
crystal 1.4 A O2 1.12 A O2 1.21 A O2- 1.33
A H2O2/O22- 1.49 A
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Naphthalene dioxygenase with indole as substrate
In E. coli, tryptophanase provides indole (3),
steps b and c are spontaneous. Naphthalene
dioxygenase is therefore enough to make indigo
(6).
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Naphthalene Ring cleavage by 1,2-dihydroxynaphtha
lene dioxygenase (NahC). The sequence of a
chromosomal 11.5 kbp fragment of Pseudomonas
stutzeri AN10 revealed nine putative genes for
naphthalene degradation, which are proposed to
catalyze six steps (NahA has 4 subunits)
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Naphthalene Ring cleavage is achieved by NahC, a
1,2-dihydroxynaphthalene dioxygenase.
What type of enzyme is NahC? The closest relative
to NahC is catechol-2,3-dioxygenase
It belongs to the extradiol dioxygenases
(cleavage outside of the ortho OH-groups), that
have a nonheme Fe2 (in rare cases Mn2) in the
active site (intradiol dioxygenases have a
nonheme Fe3).