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Microbiology 155 (2009), 3083-3091; DOI  10.1099/mic.0.030460-0
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Microbiology 155 (2009), 3083-3091; DOI  10.1099/mic.0.030460-0
© 2009 Society for General Microbiology

Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase

Jaigeeth Deveryshetty and Prashant S. Phale

Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology – Bombay, Powai, Mumbai 400 076, India

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 ~39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with Km 13.5 µM and Vmax 114 µmol 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 Ki of 116 µM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a Ki of 24 µM. 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 O2 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.

Correspondence
Prashant S. Phale
pphale{at}iitb.ac.in

Abbreviations: 2-CBADH, 2-carboxybenzaldehyde dehydrogenase; GDO, gentisic acid 1,2-dioxygenase; 2-H1NA, 2-hydroxy-1-naphthoic acid; 1-H2NA, 1-hydroxy-2-naphthoic acid; 3-H2NA, 3-hydroxy-2-naphthoic acid; 6-H2NA, 6-hydroxy-2-naphthoic acid; 1-HNDO, 1-hydroxy-2-naphthoic acid dioxygenase; P3,4DO, protocatechuate 3,4-dioxygenase; P4,5DO, protocatechuic acid 4,5-dioxygenase; SDO, salicylic acid 1,2-dioxygenase






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