Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase fromPseudomonas fluorescensIP01

Abstract

The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.Aromatic hydrocarbons are common contaminants of soil and groundwater (18). One of the most attractive means of removal of these compounds from the environment is the use of microorganisms (34). Dihydroxylation of the aromatic ring by a bacterial aromatic hydrocarbon dioxygenase is a prerequisite for subsequent oxidation of the aromatic nucleus by a ring fission dioxygenase (6). Aromatic hydrocarbon dioxygenases belong to a large family named the Rieske nonheme iron oxygenases (11). Werlen et al. delineated four dioxygenase subfamilies in this large family (the toluene/biphenyl, naphthalene, benzoate, and phthalate subfamilies) based on sequence alignment of the catalytic components (␣ subunits) (37). The toluene/biphenyl subfamily includes enzymes for the degradation of toluene, benzene, cumene (isopropylbenzene), biphenyl, and polychlorinated biphenyls (PCBs). The naphthalene subfamily consists of enzymes for the degradation of naphthalene and phenanthrene. The Rieske dioxygenases involved in bacterial hydrocarbon degradation comprise multicomponent enzyme systems (36) in which reduced pyridine nucleotide is used as the initial source of two electrons for dioxygen activation. The electrons pass through a flavin cofactor and Rieske [2Fe-2S] centers into the mononuclear iron center of the terminal Rieske nonheme iron dioxygenase component.The crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 (NphDO) has been reported previously (4,15,17), and the structure-function relationship of this enzyme has been well studied (28). NphDO is an ␣ 3 ␤ 3 hexamer, and each ␣ subunit contains a Rieske [2Fe-2S] cluster and nonheme iron coordinated by His208, His213, and Asp362. The active site iron center of one of the ␣ subunits is directly connected by hydrogen bonds through a singl...

Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source

“…However, this remains to be substantiated via cloning of NdmA and NdmB individually. Some Rieske oxygenases are known to be in hexameric a 3 b 3 configuration (Dong et al, 2005;Friemann et al, 2005;Kauppi et al, 1998;Yu et al, 2007) but only the a subunits contain the [2Fe-2S] cluster and the mononuclear ferrous iron, while the b subunits are mainly for structural purposes (Ferraro et al, 2005). The 2-oxo-1,2-dihydroquinoline 8-monooxygenase of P. putida 86 was reported to be in an a 6 configuration, determined by gel filtration chromatography (Rosche et al, 1995), but the crystal structure showed that it is actually a homotrimer (Martins et al, 2005).…”

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

“…Several three-dimensional (3D) structures of ROs are currently available (3,4,6,7,8,9,13,15,17,19,20), including a single 3D structure of KshA, namely, that of M. tuberculosis H37Rv (Rv3526; KshA H37Rv ) (2). Although the protein sequences of ROs vary considerably, their tertiary structures are very similar overall.…”

“…Characteristic for ROs, including KshA H37Rv , is the formation of trimers. These trimers either occur as ␣3 subunits (2,4,17,19) or may additionally include a smaller ␤ subunit of the oxygenase component, resulting in an (␣␤)3 enzyme complex (3,6,7,8,9,13,15,20). The active site of KshA H37Rv was shown to be composed of a ␤ sheet, including a loop region located at the entrance of the active site, flanked by two ␣ helices.…”

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

“…The 3D structure was determined by molecular replacement, using the α-subunit from naphthalene dioxygenase from Pseudomonas sp. Strain NCIB 9816-4, [6] and the β-subunit from cumene dioxygenase from P. fluorescens strain IP01, CDO-O IP01 [11] as templates. The model was refined using REFMAC [12] and COOT [13] to R and R free factors of 19.7 and 23.6 %, respectively (Fig.…”