Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase

Abstract

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe 2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7-1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe 2+ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe 2+, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe 2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O 2 -dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.dioxygen activation | non-heme iron | amino acid degradation | Pseudomonas putida | alkaptonuria H omogentisate (2,5-dihydroxyphenylacetate, HG) is the central metabolite in the degradation pathways of phenylalanine and tyrosine in aerobic organisms ranging from soil bacteria like Pseudomonas putida to man (1, 2). Oxidative ring cleavage is catalyzed by homogentisate 1,2-dioxygenase (HGDO), which incorporates the atoms of molecular oxygen into HG to produce maleylacetoactate (1, 3-6). A deficiency of HGDO is known to cause the autosomal recessive disorder alkaptonuria in humans, the first genetic defect to be recognized as such (6-8). The crystal structure of HGDO from man (HGDO Hs ) revealed that the enzyme belongs to the cupin fold type of nonheme iron-dependent dioxygenases, forming a homohexamer consisting of a dimer of trimers (6). The active site of HGDO Hs employs the 2-His-1-carboxylate facial triad to bind an essential Fe 2+ ion with a distorted square-pyramidal arrangement (6). The facial triad motif is found in various nonhomologous types of nonheme Fe 2+ -dependent enzymes and commonly serves to control the reactivity of the Fe 2+ site and to activate an aromatic or aliphatic substrate together with dioxygen (9) and is also featured in extradiol-type dioxygenases such as 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) and homoprotocatechuate 2,3...

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

“…Very recently, three reaction intermediates were also found in an in crystallo study of homogentisate dioxygenase (HGD) [47]. Homogentisate is an isomer of HPCA, differing in having the two hydroxyl groups para to each other rather than ortho.…”

O2 Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162

“…Salicylate 1,2-dioxygenase (SDO) belongs to the family of homo-tetrameric class III ring fission dioxygenases, together with 3-hydroxyanthranilate-3,4-dioxygenase, 1-hydroxy-2-naphthoate dioxygenase, gentisate dioxygenase, and homogentisate dioxygenase (HGDO). 5- 13 The SDO enzyme utilizes O 2 to cleave the aromatic ring of the substrate salicylate (Sal) and inserts two oxygen atoms into the product (Scheme 1).…”

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

“…A powerful extension of this approach involves performing catalysis in enzyme crystals to trap reactive species for characterization using X-ray crystallography, which has been successfully applied to a few systems (27)(28)(29)(30)(31). This approach was used by our laboratory to identify Significance Vast quantities of aromatic compounds enter the environment due to the natural breakdown of lignin as well as industrial and agricultural pollution.…”