Pergamon Chemosphere, Vol. 38, No. 6, pp. 1331-1337,1999 © 1999Elsevier Science Ltd.All rightsreserved 0045-6535/99/$ - see frontmatter Pll: S0045-6535(98)00534-7 CHARACTERIZATION OF GENES FOR ENZYMES INVOLVED IN THE PHENANTHRENE DEGRADATION IN NOCARDIOIDES SP. KP7 Atsushi Saito, Tokuro Iwabuchi, Shigeaki Harayama* Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan Abstract : The nucleotide sequence of the gene cluster, phdEFABGHCD, encoding enzymes responsible for the transformation of phenanthrene to l-hydroxy-2-naphthoate in Nocardioides sp. strain KP7 was determined. This gene cluster, which may constitute a single operon, resided at 6.1-kb downstream of the phdlJK gene cluster encoding the enzymes for the transformation of 1-hydroxy-2-naphthoate to o-phthalate. In general, the phd products exhibited moderate degrees of homology with isofunctional enzymes found in pathways for the degradation of other aromatic compounds. Remarkably, the phdC gene product had features of the [3Fe-4S] type ferredoxin, which has not been found so far as a component of the ring- hydroxylating dioxygenase. Escherichia coli carrying the genes for phenanthrene dioxygenase, phdABCD, was capable to oxidize phenanthrene. INTRODUCTION Studies on the microbial degradation of naphthalene and phenanthrene have revealed particular aspects of the metabolism of polycyclic aromatic hydrocarbons (PAHs) [12,1. The biodegradation of PAHs is generally initiated by the reaction catalyzed by a multicomponent ring-hydroxylating dioxygenase. At this step, both atoms of molecular oxygen are incorporated into the aromatic nucleus 11]. In bacteria, phenanthrene is degraded via l-hydroxy-2-naphthoate to tricarboxylic acid cycle intermediates. The enzymatic steps involved in the conversion of phenanthrene to 1-hydroxy-2-naphthoate have been revealed to be very similar to those involved in the transformation of naphthalene to salicylate [3, 4]. Nocardioides sp. strain KP7 [5] is a high G+C content Gram-positive bacterium, which is able to grow by using phenanthrene as sole carbon and energy sources. Pbenanthrene is degraded by this bacterium via 1-hydroxy-2-naphthoate and o-phthalate. Recently, the nucleotide sequence of the genes encoding 1- hydroxy-2-naphthoate dioxygenase (p/u//), trans-2'-carboxybenzalpyruvate hydratase-aldolase (phdJ), and 2-carboxybenzaldehyde dehydrogenase (phdK) in this organism was determined and the biochemical properties of these enzymes were characterized [6-8]. In this report, we describe the structure of the whole phd gene cluster and the relationships of the phd genes with the counterparts in pathways for the degradation of other aromatic hydrocarbons. We also report 1331