Crystal structures of substrate free and complex forms of reactivated BphC, an extradiol type ring-cleavage dioxygenase

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Abstract

BphC derived from Pseudomonas sp. strain KKS102, an extradiol type catecholic dioxygenase, is a non-heam iron-containing enzyme, playing an important role in the degradation of biphenyl/PCB (Poly Chlorinated Biphenyls) in the microbe. Although we had earlier solved the crystal structure of KKS102 BphC, it was the inactive form with Fe(III) in the active site. In order to determine the active form structure, BphC was re-activated by anaerobic incubation with Fe(II) and ascorbate, and crystallized anaerobically. The crystal structures of activated BphC and its substrate complex (E·S complex) were determined at 2.0 Å resolution under cryogenic condition. In addition, crystal structures of unactivated BphC in substrate free and complex forms were also re-determined. Comparison of activated and unactivated E·S complexes reveals that the orientation of the bound substrate in the active site is significantly different between the two. The structural comparison of the substrate free and complex forms of activated BphC show certain small conformational shifts around the active site upon substrate binding. As a result of the conformational shifts, His194, which has been suggested as the catalytic base, takes part in a weak hydrogen bond with hydroxyl group of the substrate.

Introduction

Extradiol type catecholic ring cleavage dioxygenases (hereafter extradiol type dioxygenase) are key enzymes in the degradation pathways of aromatic compounds such as benzene, toluene and polychlorinated biphenyls (PCBs). To date, various types of extradiol type dioxygenases have been cloned from microorganisms such as Pseudomonads [1]. As shown in Fig. 1, these enzymes catalyze the addition of two atomic oxygens to the catechol ring of the substrate resulting in the cleavage of the catechol ring. These extradiol type dioxygenases typically contain one non-heme iron (Fe(II)) in their active site but have also been shown to be active with Mn(II) [2].

To date, three classes of extradiol type (catecholic) dioxygenases have been identified, classes I, II and III [3]. Comparison of the primary structures between classes I and II enzymes have revealed that the two classes of enzymes have a common ancestor, the class II enzymes having evolved from a class I enzyme through gene duplication [3], [4]. As a result, the enzymes are composed of two domains and each has a similar structural topology [5], [6], [7], [8].

The KKS102 BphC enzyme (hereafter BphC) is composed of eight identical subunits, each of which is related by the 422-point group symmetry. Each subunit of the BphC enzyme consists of two domains, domain 1 and domain 2. Each domain consists of two repetitions of a βαβββ motif, thus, the subunit is composed of four repetitions of the βαβββ motifs [5], [6]. The Fe(II) ion, which is essential for the enzymatic reaction, is located inside the barrel-like structure of domain 2, and coordinated by His145, His209 and Glu260. Similar structures have also been reported for the BphC isolated from P. cepacia [7], metapyrocatechase from P. putida mt-2 [8] and recently for the homoprotocatechuate 2,3-dioxygenases containing Fe2+ and Mn2+ ions [9]. The BphC from P. cepacia, closely related to the KKS102 BphC, has been solved in Fe(II) containing active form [7] and in three complexes with alternative substrate molecules [10].

To gain insights into the catalytic mechanism of the class II extradiol type dioxygenases, we previously determined the crystal structures of 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC) derived from Pseudomonas sp. strain KKS102 [11], in the substrate free form and in a ferric complex with its substrate, 2,3-dihydroxybiphenyl (2,3-DHBP), which was inactive [5], [6]. The iron center of the substrate free form of BphC can be described as a five-coordinate square-pyramidal center with one axial histidine (His145) and four basal ligands (His209, Glu260 and two water molecules) [5], [6], [7]. Upon binding the substrate, 2,3-DHBP, the two water molecules are dissociated from the iron coordination sphere, while two hydroxyl groups of the substrate coordinate to the iron, resulting in a octahedral iron center with one vacant site in the equatorial plane (or a trigonal bipyramidal iron center [6]). The equatorial ligands are His145, Glu260 and a hydroxyl group of the substrate; the axial ligands are His209 and another hydroxyl group of the substrate. The vacant site has been proposed as the binding site for a dioxygen [6].

In addition to our crystallographic analysis, many mutant proteins have been prepared and their kinetic parameters and crystal structures have been determined (Nishizaki et al., in preparation). These analyses have revealed that (1) His194 is indispensable for the catalytic activity, and (2) the shape of the substrate-binding pocket and its hydrophobic character are important for substrate binding [12].

However, in our earlier studies, all the experiments were carried out under aerobic conditions. Consequently the crystals used in those studies were mainly composed of inactive proteins. Our previously determined structures of BphC and its substrate complex may differ from the active Fe(II) containing structures. Moreover to deduce more detailed mechanistic insights, precise geometry of the active site is required with BphC in the active form. To that end we have determined the crystal structures of substrate free and complex forms using BphC in its active form at 2.0 Å resolution and compared them to the corresponding structures obtained with inactive enzyme. The comparison reveals that the orientation of the bound substrate in activated BphC is significantly different from that of unactivated BphC. The analysis of the crystal structures of substrate free and complex forms of activated BphC reveals small conformational shifts of residues around the active site upon substrate binding.

Finally one reservation has been made as to the relevance of the structures presented here with respect to the active site of the enzyme. In this study we did not determine (1) the total iron content in the enzyme either as isolated or after activation with ascorbate and Fe(II)SO4, (2) the redox state of the active site iron in either form of the enzyme, (3) or conclusively establish the maximum specific activity attainable in solution or crystalline conditions. Thus, in the strict sense, the activated enzyme, which has a substantially higher enzyme activity than oxidized enzyme, cannot be regarded as the “fully” activated enzyme. The study to obtain the fully activated enzyme is under way.

Section snippets

Expression and purification of BphC

The BphC was overexpressed from a pHNA2 vector in E. coli strain MV1190 as described elsewhere [13]. The cells were disrupted by sonication to obtain cell extract. Nucleic acids and membrane fraction in the cell extract were removed using a centrifuge yielding cytoplasmic fraction. The cytoplasmic fraction whose pH was adjusted to 7.5 was applied to an anion-exchange column (DEAE-TOYOPEARL 650S, (TOSOH) 30×170 mm) equilibrated with 25 mM of phosphate buffer at pH 7.5 (hereafter, the

Reactivation and crystallization of BphC

Table 1 clearly shows that incubation with both ferrous ion and ascorbate are required to increase enzyme activity of BphC. BphC is reactivated rapidly by anaerobic mixing with the reactivation buffer. Even though the activity reaches a maximum in approximately 10 min, an incubation period of about 2 h has been used in the present protocol. However, the maximum activities of different batches of purified sample were not consistent, suggesting that there could be other factors (stability of the

Accession numbers

The coordinates and structure factors for the BphC structures have been deposited with the Protein Data Bank with the accession codes 1EIL (activated BphC in substrate free form) 1EIM (activated BphC in complex with 2,3-DHBP), 1EIQ (unactivated BphC in substrate free form), and 1EIR (unactivated BphC in complex with 2,3-DHBP).

Acknowledgements

We thank Dr. T. Nonaka of the present group for crystallographic discussions. We also thank Drs. M. Suzuki and N. Igarashi of PF for their assistance in anaerobic data collection of activated BphC. This study was partly supported by the Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) in Japan, the Sakabe project at the TARA (Tsukuba Advanced Research Alliance) center, University of Tsukuba, Japan and Grants-in-Aid for Scientific Research from the Ministry of

References (29)

  • M. Fukuda

    Curr. Opinion Biotechnol.

    (1993)
  • A.K. Whiting et al.

    Biochemistry

    (1996)
  • E.L. Spence et al.

    J. Bacteriol.

    (1996)
  • L.D. Eltis et al.

    J. Bacteriol.

    (1996)
  • K. Sugiyama et al.

    Porc. Japan Acad.

    (1995)
  • T. Senda et al.

    J. Mol. Biol.

    (1996)
  • S. Han et al.

    Science

    (1995)
  • A. Kita et al.

    Structure

    (1999)
  • M.W. Vetling et al.

    J. Inorg. Biochem.

    (1999)
  • F.H. Vaillancourt et al.

    J. Biol. Chem.

    (1998)
  • K. Kimbara et al.

    J. Bacteriol.

    (1989)
  • T. Senda et al.
  • K. Sugiyama et al.

    Proteins

    Struct. Funct. Genet.

    (1995)
  • N. Sakabe

    Nucl. Instrum. Methods

    (1991)
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