Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

Abstract

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The K m for dioxygen was 1280 ؎ 70 M, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. K m and K d for DHB were 22 ؎ 2 and 8 ؎ 1 M, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.The microbial degradation of aromatic compounds constitutes an essential link in the global carbon cycle. The aerobic degradation of aromatic compounds such as toluene, naphthalene, and biphenyl generally proceeds via a catecholic catabolite with hydroxyl substituents on two adjacent carbon atoms. This catecholic compound is cleaved by a dioxygenase from one of two very different classes. Intradiol dioxygenases utilize non-heme ferric iron to cleave the aromatic nucleus ortho to (between) the hydroxyl substituents whereas extradiol dioxygenases utilize non-heme ferrous iron to cleave the aromatic nucleus meta (adjacent) to the hydroxyl substituents. The mechanism of intradiol dioxygenases is better understood due to their greater stability, favorable properties for spectroscopic examination, and the accessibility of catalytic intermediates (1, 2). Interest in extradiol dioxygenases is nonetheless considerable, not only because of their general metabolic significance and catalytic properties, but also because of the potential exploitation of these enzymes in the degradation of environmental pollutants such as polychlorinated biphenyls.2,3-Dihydroxybiphenyl 1,2-dioxygenase (DHBD) 1 is a component of the aerobic biphenyl degradation pathway of a number of microorganisms and cleaves 2,3-dihydroxybiphenyl (DHB) in an extradiol fashion as shown in Scheme 1. Crystallographic studies of DHBD from Burkholderia cepacia LB400 (3) and Pseudomonas...

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

“…An alternative explanation could be that the substrate binds to a second site, maybe in the N-terminal domain, and this has a negative allosteric effect on activity. However, substitutions V175D and Q198D, which significantly altered the K m of the enzyme, also altered sensitivity to substrate inhibition (Table 2), thus indicating that inhibition is apparently exerted through the same binding site, as previously suggested by kinetic analysis of the DHBD from LB400 (Vaillancourt et al, 1998). A possible mechanism for the substrate inhibition would be that high concentration of the substrate at the 'entranceway' might jam the enzyme, thus preventing proper fitting of the substrate in the active site, or release of the product after catalysis.…”

SyrB2 in syringomycin E biosynthesis is a nonheme Fe II α-ketoglutarate- and O 2 -dependent halogenase

“…Chromatography was performed by using an Ä KTA Explorer 100 (GE Healthcare) configured to maintain an anaerobic atmosphere during purification, as described in ref. 15. Buffers were sparged with Argon and equilibrated in the glove box for 24 h before use.…”

Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation

“…Steady-state Kinetic Measurements and Data Analysis-Ring-cleaving activity was measured by following the consumption of dioxygen using a Clark-type polarographic electrode (Yellow Springs Instrument Co. model 5301, Yellow Springs, OH) as previously described (17). All experiments were performed using phosphate buffer, pH 7.0, I ϭ 0.1 M, 25.0 Ϯ 0.1°C (290 M dissolved O 2 ) unless otherwise stated.…”

“…The cell pellet originating from a 4-liter culture was resuspended in 10 mM Tris, 10% glycerol, pH 7.5. Subsequent manipulations were performed anaerobically as described previously (17). The cells were disrupted by three successive passages through a French press (Spectronic Instruments Inc., Rochester, NY) operated at a pressure of 20,000 p.s.i.…”

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