Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally

Abstract

The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4-chlorinated ring (97%) of 2,5,4-trichlorobiphenyl and introduced molecular oxygen at the 2,3 position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O 2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.Recent studies revealed that oxygenases involved in the initial oxidation of aromatic hydrocarbons are multicomponent enzymes and that their corresponding subunits show various degrees of homology (14,26,27,31). This implies that soil bacteria have adaptively evolved, by modifying key enzymes, to utilize a variety of aromatic compounds which are present in the environment. Biphenyl-utilizing bacteria are ubiquitously distributed and can be isolated from various environmental samples (3,8,29) including intestine of termite (5), suggesting that they are involved in the degradation of plant lignin at the final stage, together with other aromatic degraders (24). Biphenyl-utilizing bacteria have been extensively studied with respect to the degradation of polychlorinated biphenyls (PCB), which are known to be serious environmental pollutants. These studies revealed considerable differences in the congener selectivity patterns and in the range of activity of various PCBdegrading bacteria (9). It was also demonstrated that both the relative rates of primary degradation of PCB and the choice of the ring attacked were dependent on the bacterial strains (1, 2, 4, 16). Pseudomonas pseudoalcaligenes KF707 was isolated from soil near a biphenyl-producing factory in Kitakyushu, Japan (12,15). This strain showed a narrow range of degradable PCB congeners (7, 17). Pseudomonas sp. strain LB400 (referred to as Pseudomonas cepacia LB400 in a recent publication [19]) was isolated from a PCB...

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

“…Differences in CB dioxygenation between BDO-H09 and -H03 clearly demonstrate the importance of region αE, in agreement with findings made in the LB400\KF707 system (Erickson & Mondello, 1993 ;Kimura et al, 1997 ;Mondello et al, 1997). However, the difference in CB dioxygenation between BDO-H09 and BDO-LB400 was not expected on the basis of those studies.…”

“…As regions αG and αH have been shown to be of minor importance, this result highlights a significant effect of region αF. Previously, Kimura et al (1997) and Mondello et al (1997) identified a major contribution of aa 377 (LB400 numbering) on substrate specificity. This is one of the three amino acid differences between regions αF of the LB400 and P6 subunits.…”

“…Electrophoresis was carried out in 0n1 % SDS\12 % polyacrylamide gels as described previously (Hofer et al, 1993). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al, 1989 deduced from the crystal structure of a naphthalene dioxygenase (Kauppi et al, 1998 ;Carredano et al, 2000) and from functional studies on BDO variants (Erickson & Mondello, 1993 ;Kimura et al, 1997 ;Mondello et al, 1997). Hatched and white boxes represent the catalytic and Rieske domains, respectively.…”

“…A strategy to overcome this difficulty is the generation of enzymes with novel substrate specificities by protein engineering. Sequence elements of biphenyl dioxygenases (BDOs) that significantly influence substrate specificity have been identified by exchanges of M. Zielinski, S. Backhaus and B. Hofer entire subunits or parts of α subunits (Erickson & Mondello, 1993 ;Furukawa et al, 1994 ;Hirose et al, 1994 ;Kimura et al, 1997 ;Mondello et al, 1997). These approaches used segments of the bph-encoded large subunits of Burkholderia sp.…”

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

“…Previous studies with class II ARHDO systems have demonstrated that the major determinants of the fundamental catalytic properties such as substrate and product spectra reside within the C-terminal part of the large or a-subunit (Erickson & Mondello, 1993;Kimura et al, 1997;Mondello et al, 1997;Beil et al, 1998;Zielinski et al, 2002), although the small or b-subunit has occasionally been reported to exert some influence on these properties (Furukawa et al, 1994;Hurtubise et al, 1998). Recently, it has been shown that the C-terminal 60 aa of the a-subunit are of minor importance for these properties (Zielinski et al,…”