title: Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase.
abstract: Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the 'phthalic acid' route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of approximately 39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with K(m) 13.5 microM and V(max) 114 micromol min(-1) mg(-1). 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K(i) of 116 microM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K(i) of 24 microM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O(2) into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradiol-type ring-cleaving dioxygenase.
authors: Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology - Bombay, Powai, Mumbai 400 076, India.
authors: Jaigeeth
authors: J
authors: Deveryshetty
authors: Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology - Bombay, Powai, Mumbai 400 076, India.
authors: Prashant S
authors: PS
authors: Phale
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D001711:Biotransformation; D002614:Chelating Agents / Q000378:metabolism; D049308:Dioxygenases / Q000302:isolation & purification / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D004533:Egtazic Acid / Q000378:metabolism; D004789:Enzyme Activation; D053858:Metabolic Networks and Pathways; D009284:Naphthols / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D009284:Naphthols; D010616:Phenanthrenes; C031181:phenanthrene; D004533:Egtazic Acid; D004492:Edetic Acid; D049308:Dioxygenases; C050175:1-hydroxy-2-naphthoic acid