Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3

Abstract

Hydroquinone-1,2-dioxygenase, an enzyme involved in the degradation of alkylphenols in Sphingomonas sp. strain TTNP3 was purified to apparent homogeneity. The extradiol dioxygenase catalyzed the ring fission of hydroquinone to 4-hydroxymuconic semialdehyde and the degradation of chlorinated and several alkylated hydroquinones. The activity of 1 mg of the purified enzyme with unsubstituted hydroquinone was 6.1 μmol per minute, the apparent Km 2.2 μM. ICP-MS analysis revealed an iron content of 1.4 moles per mole enzyme. The enzyme lost activity upon exposure to oxygen, but could be reactivated by Fe(II) in presence of ascorbate. SDS-PAGE analysis of the purified enzyme yielded two bands of an apparent size of 38 kDa and 19 kDa, respectively. Data from MALDI-TOF analyses of peptides of the respective bands matched with the deduced amino acid sequences of two neighboring open reading frames found in genomic DNA of Sphingomonas sp strain TTNP3. The deduced amino acid sequences showed 62% and 47% identity to the large and small subunit of hydroquinone dioxygenase from Pseudomonas fluorescens strain ACB, respectively. This heterotetrameric enzyme is the first of its kind found in a strain of the genus Sphingomonas sensu latu.

Figure 1

SDS-PAGE of HQDO from Sphingomonas sp. strain TTNP3. Lane A, marker proteins: lane B, crude cell extract; lane C, ammonium sulfate fractionation supernatant; lane D, phenyl-Sepharose pool; lane E, DEAE pool; lane F; MonoQ pool.

Figure 2

A, GC-MS total ion chromatogram of the trimethylsilylated ring cleavage product of hydroquinone; B, mass spectrum of peak 1b from Figure 2A; C, GC-MS total ion chromatogram of the trimethylsilylated ring cleavage product of 2-methylhydroquinone; D, mass spectrum of the product peak 2c from Figure 2C.

Figure 3

Amino acid sequences deduced from open reading frames found in a part of genomic DNA from Sphingomonas sp. strain TTNP3. The amino acids marked with asterisks were identified by nanoHPLC-nanoESI-MS/MS (from residues 175 to 194, two peptides were matched, one ranging from 175 to 184, the other one from 185 to 194).

Figure 4

Phylogenetic trees of the sequences of HqdA (A) and HqdB (B) and the respective homolog sequences from Burkholderia sp. 383 (gi 78063587 and gi 78063586), Burkholderia sp. CCGE1002 (gi 295680998 and gi 295680997), Burkholderia sp. H160 (gi 209517843 and gi 209517844), Burkholderia ambifaria AMMD (gi 115359956 and gi 115359957), Burkholderia ambifaria IOP40-10 (gi 170700037 and gi 170700038), Burkholderia ambifaria MC40-6 (gi 172062406 and gi 172062407), Burkholderia ambifaria MEX-5 (gi 171319707 and gi 171319708), Burkholderia cenocepacia HI2424 (gi 116691528 and gi 116691529), Burkholderia cenocepacia J2315 (gi 206562327 and gi 206562328), Burkholderia multivorans ATCC 17616 (gi 161523095 and gi 161523094) Burkholderia multivorans CGD1 (gi 221212137 and gi 221212136), Burkholderia multivorans CGD2M (gi 221199017 and gi 221199016), Burkholderia phymatum STM815 (gi 186470422 and gi 186470423), Photorhabdus luminescens subsp. laumondii TTO1 (gi 37524165 and gi 37524166), Pseudomonas aeruginosa PA7 (gi 152988009 and gi 152987326), Pseudomonas fluorescens ACB (gi 182374631 and gi 182374632), Pseudomonas putida (gi 224460045 and gi 260103908), Pseudomonas sp. 1-7 (gi 284176971 and gi 284176972), Pseudomonas sp. NyZ402 (gi 269854714 and gi 269854713), Pseudomonas sp. WBC-3 (gi 156129389 and gi 156129388) and Variovorax paradoxus S110(gi 239820773 and gi 239820774). Sequences were retrieved from NCBI via BLAST search, subsequently aligned using ClustalX 2 and rendered using Treeview 1.6.6.