Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

• Hydroxamic acid inhibitor D13 developed for protocatechuate 3,4-dioxygenase.
• Treatment of Rhodococcus jostii with D13 leads to formation of protocatechuic acid.
• Treatment of R. jostii RHA1 with disulfiram leads to formation of 4-carboxymuconolactone.
• Treatment of P. fluorescens with disulfiram generates glycine betaine aldehyde.

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15 μM) and D3 for MhpB (IC50 110 μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1 mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.

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