Crystal Structure and Resonance Raman Studies of Protocatechuate 3,4- Dioxygenase Complexed with 3,4-Dihydroxyphenylacetate^,

Abstract

The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA. Fe(III) chelate complex. 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

“…The slowest phase has an RRT very similar to k cat from steady-state experiments (0.18 s −1 at 4°C). In all previous studies, we and others have found that product release is the rate-limiting step for IDOs, and the preceding ringopening step is irreversible (12,24,25,35). This irreversible step allows the slowest RRT to be associated with the rate constant for a specific step in the reaction, presumably product release.…”

“…In 3,4-PCD this coordination mode comes about as a result of the rearrangement described above and is stabilized by interaction with second sphere conserved residues Q477 and R457. As shown in Axial binding of catecholic substrates is a characteristic of all classes of IDOs (20,23,35,42). The alternative equatorial 4FC binding mode provides insights into the driving force behind the substrate binding-mediated dissociation of Y447.…”

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

“…The refinement of the enzyme-DHB complex is described elsewhere. 2 The procedures were similar, but the substrate-binding site was fully occupied by DHB so that the use of two models of the active site was not necessary.…”

“…Consistent with previous studies (13), the apparent specificity of DHBD in air-saturated buffer is 350-fold higher for DHB than for catechol. Although the K mO 2 of DHBD in the presence of catechol could not be evaluated, at a concentration of 1275 M catechol, the apparent constant, K mO 2 app was 1000 Ϯ 160 M.…”

“…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.…”