Multiple alignment of 2,4-dioxygenases, various hydrolases, and some cofactor-free haloperoxidases in the region of the potential catalytic nucleophile of the enzymes. The alignments are based on database searches and binary alignments run with the program BLAST (1) and have been manually realigned. Abbreviations (see also the legend to Fig. 1): BphD, P. putida KF715 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (18) (the amino acid sequences of BphD from strain KF715 and from Pseudomonas sp. strain LB400 [22] show 95% identity); TodF, P. putida F1 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (37); DmpD, Pseudomonas sp. CF600 2-hydroxymuconic semialdehyde hydrolase (41); Lip3, Moraxella sp. strain TA144 lipase 3 (13); CpoP, Pseudomonas pyrrocinia chloroperoxidase (62); LinB, Sphingomonas (formerly Pseudomonas) paucimobilis 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase (38); DhaA, Rhodococcus rhodochrous NCIMB 13064 1-chloroalkane dehalogenase (30); DehH1, Moraxella sp. strain B haloacetate dehalogenase (27); EchA, Agrobacterium radiobacter AD1 epoxide hydrolase (49); rsEH, soluble epoxide hydrolase from rat liver (28); hPL, human pancreatic lipase (59). The structures of BpoA2 (19), DhlA (15, 56, 57), EchA (39), and hPL (59) (the abbreviations are underlined in the figure) have been solved by X-ray diffraction. The putative catalytic nucleophilic residues are underlined and marked by a star. S93 and D120 of Qdo and the catalytic triad acid D176 of hPL are marked by arrows. Amino acid residues conserved in at least half of the aligned sequences are in boldface.

SDS-PAGE of recombinant His₆-tagged Qdo proteins. (a) His₆Qdo variants purified by metal chelate affinity chromatography. Lanes: 2, recombinant Qdo; 3, QdoD120A; 4, QdoD219A; 5, QdoH244A; 7, QdoS95A; 8, QdoS95C. (b) Crude extracts of E. coli clones expressing recombinant His₆-tagged Qdo (lane 2) and of clones supposed to express His₆-tagged QdoS93A (lane 3) and QdoS213A (lane 4). The molecular masses of the marker proteins (lanes 1, 6, and 9 in panel a and lanes 1 and 5 in panel b) are 97.2, 66.2, 55, 42.7, 40, and 31 kDa.

Multiple amino acid sequence alignment of Qdo, Hod, and some hydrolases and cofactor-free haloperoxidases performed with the program CLUSTAL W (55), and prediction of secondary-structure elements for Qdo and DhlA. Abbreviations: Qdo, P. putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (35; EMBL accession no. Y14779); Hod, A. ilicis Rü61a 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (35; EMBL accession no. Y14778); Tpes, P. putida atropinesterase (20); CpoL, Streptomyces lividans TK64 chloroperoxidase (4); BpoA2, Streptomyces aureofaciens ATCC 10762 bromoperoxidase (45); XylF, P. putida 2-hydroxymuconic semialdehyde hydrolase (23; the sequence has been corrected in the N-terminal region according to reference 12; see also reference 22); DhlA, Xanthobacter autotrophicus GJ10 haloalkane dehalogenase (26). The numbering does not refer to any of the sequences. Presumed triad residues are marked with a star and printed in boldface. Conserved amino acid motifs surrounding the catalytic nucleophile and the catalytic histidine residue of the α/β hydrolase-fold enzymes are enclosed in a box. S93, D120, and S213 of Qdo are marked by an arrow. The first line at the bottom of the sequences shows the secondary-structure elements of Qdo as calculated by the program PredictProtein (50). The second and third lines indicate the secondary-structure elements of DhlA as predicted (line 2) and as deduced from the X-ray analyses of DhlA (56) (line 3). Hatched and open boxes indicate β-sheets and α-helices, respectively. The α-helices of DhlA are numbered according to the numbering scheme of Verschueren et al. (56). Note that the Qdo sequence (35) has been corrected in the C-terminal region (FLQA instead of FLHGLSTCNHELKR) and at position 98 (C instead of V). Qdo thus consists of 264 amino acids, its calculated molecular mass is 30,347 Da, and its calculated isoelectric point is pH 5.43.

Multiple alignment of 2,4-dioxygenases, various hydrolases, and some cofactor-free haloperoxidases in the region of the potential catalytic aspartate and histidine residues of the enzymes. The alignments are based on database searches and binary alignments run with the program BLAST (1) and have been manually realigned. Abbreviations are given in the legends to Fig. 1 and 2. The structures of BpoA2 (19), DhlA (15, 56, 57), and EchA (39) (abbreviations are underlined in the figure) have been solved by X-ray diffraction. The putative triad histidine residues are underlined and marked by a star. The putative acidic residues of the catalytic triad are underlined. S213 and D219 of Qdo are marked by arrows. Amino acid residues conserved in at least half of the aligned sequences are in boldface.

Tentative hypothesis for the mechanism of Qdo-catalyzed 2,4-dioxygenolysis of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilic acid (for a detailed explanation, see the text). [Ser-O⁻], deprotonated serine residue of Qdo; [His-H⁺], protonated histidine residue of Qdo.