Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

Abstract

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysenedegrading organism Sphingomonas sp. strain CHY-1 were investigated. [14 C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1 a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

A New Classification System for Bacterial Rieske Non-Heme Iron Aromatic Ring-Hydroxylating Oxygenases

“…[36], S. yanoikuyae B1 (BphA1c and XylX) [40,41] and Sphingobium sp. CHY-1 (PhnA1a and PhnA1b) [42] were reported to have the same gene arrangement with 63-97% sequence identity when compared to F199. Consequently, they were classified as the same types with those from F199; type II for XylX (B1), type IIIαβ for AhdA1 [a,b] (P2), PhnA1a (CHY-1) and type IV for BphA1c (B1), AhdA1 [c-e] (P2) and PhnA1b (CHY-1).…”

The catalytic pocket of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

“…Several bacteria were found to degrade PAHs but only a few have been reported to attack four and five ring PAHs [2,3]. In Sphingomonas strain CHY-1 a single RHD has been shown to be responsible for the oxidation of a wide range of PAHs [4]. This dioxygenase consists of three components, a NADH-dependent reductase (PhnA4), a ferredoxin containing a Rieske type [2Fe-2S] cluster (PhnA3), and a terminal oxygenase, PhnI, containing both a mononuclear iron [Fe 2+ ] and a [2Fe-2S] Rieske cluster [5].…”

Soil properties and microbial ecology of a paddy field after repeated applications of domestic and industrial sewage sludges

“…The genus Mycobacterium belongs to the Actinobacteria, is a Gram-positive heterotrophic bacterium, and has been reported to be capable of degrading a wide range of PAHs, not only high-molecular-weight but also low-molecular-weight PAHs (Wick et al 2002;Zeng et al 2010). Other genera (Sphingomonas and Nocardioides) have also been reported to be involved in PAH degradation (Saito et al 2000;Demaneche et al 2004;Schippers et al 2005;Desai et al 2008;Zeinali et al 2008). The changes in the composition, structure, and diversity of the soil microbial community may result from the combined effects of soil quality, nutrient content, and various pollutants that have accumulated in the soil.…”