Structural Basis of Proteolytic Activation of l-Phenylalanine Oxidase from Pseudomonas sp. P-501

Abstract

The mature form of L-phenylalanine oxidase (PAOpt) from Pseudomonas sp. P-501 was generated and activated by the proteolytic cleavage of a noncatalytic proenzyme (proPAO). The crystal structures of proPAO, PAOpt, and the PAOpt-o-amino benzoate (AB) complex were determined at 1.7, 1.25, and 1.35 Å resolutions, respectively. The structure of proPAO suggests that the prosequence peptide of proPAO occupies the funnel (pathway) of the substrate amino acid from the outside of the protein to the interior flavin ring, whereas the funnel is closed with the hydrophobic residues at its vestibule in both PAOpt and the PAOpt-AB complex. All three structures have an oxygen channel that is open to the surface of the protein from the flavin ring. These results suggest that structural changes were induced by proteolysis; that is, the proteolysis of proPAO removes the prosequence and closes the funnel to keep the active site hydrophobic but keeps the oxygen channel open. The possibility that an interaction of the hydrophobic side chain of substrate with the residues of the vestibule region may open the funnel as a putative amino acid channel is discussed.L-Phenylalanine oxidase (EC 1.13.12.9: PAO) from Pseudomonas sp. P-501 catalyzes both the oxidative deamination and oxygenative decarboxylation of L-Phe, L-Tyr, L-Trp, and L-Met as a substrate (1-3). The enzyme was screened from bacteria in soil for the clinical determination of L-Phe (4, 5). The enzyme has 2 mol of noncovalent FAD (2) and consists of 2 mol each of ␣ and ␤ subunits (6). Of the above-mentioned amino acids, L-Phe is mainly oxygenated, and L-Met is mainly oxidized by PAO. Kinetic and resonance Raman studies of the enzyme revealed that PAO catalyzes the reaction by the ping-pong type mechanism (7) and that the catalytically important species is the purple intermediate, which consists of a reduced enzyme and an imino acid derived from an amino acid substrate (8). We determined the nucleotide sequence of DNA encoding the enzyme protein (9) (see Fig. 1). The open reading frame encoding a single polypeptide is arranged in the order of initiator Met, a prosequence (14 amino acid residues long), an ␣ subunit (92 amino acid residues long), a dipeptide, and a ␤ subunit (605 amino acid residues long). The polypeptide with the prosequence (proPAO) was successfully expressed in Escherichia coli (9). The purified proPAO contains FAD, but the proenzyme does not show any catalytic activity. Protease (Pronase-trypsin mixture) treatment of proPAO initially removes only the Ile 107 -Lys 108 dipeptide from proPAO, but the removal of this peptide does not lead to the expression of enzyme activity. Further treatment with the protease removes the prosequence from proPAO, and the resultant protein shows activity. The Pronasetrypsin-treated proPAO (PAOpt) 3 showed spectral and kinetic properties comparable with the native enzyme.Several secreted enzymes are expressed as a proform. The prosequence usually acts as an intramolecular chaperone and/or an inhibitor of catalytic activity. The bes...

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

“…Experiments in the presence of D-Trp revealed a moderate VioA inhibition (72% relative activity, 10 mM D-Trp). In the present literature, citrate and o-aminobenzoate have been described as efficient inhibitors of LAAOs (21,25,26). In agreement with these distantly related investigations, a relative activity of 15% in the presence of 10 mM citrate was observed.…”

Mutational and crystallographic analysis of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

“…PAO from Pseudomonas sp. P‐501 [8] shows a relatively low structural similarity ( Z score = 33.1 and RMSD = 2.8 Å for 497 Cα atoms), and it was the 5th hit of the structural similarity search. l ‐AAO/MOG is also structurally similar to human monoamine oxidase A (7th hit, Z score = 29.8 and RMSD = 2.6 Å for 404 Cα atoms) as well as snake venom l ‐AAOs (Fig.…”

“…X‐119‐6 [7], and l ‐phenylalanine oxidase (EC 1.13.12.9, PAO) from Pseudomonas sp. P‐501 [8], have been also available. The crystal structure of tryptophan‐2‐monooxygenase (EC 1.13.12.3, TMO) from Pseudomonas savastanoi has recently been reported [9].…”

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

“…In addition to the structure of Calloselasma rhodostoma LAAO, crystal structures have also been determined of the enzymes from the venom of Agkistrodon halys pallas [51] and from bacterial sources including Rhodococcus opacus [52] and Streptomyces species [34], where the enzyme has been called l ‐glutamate oxidase, and Pseudomonas species, where the enzyme has been called l ‐phenylalanine oxidase [53]. The structures of snake venom LAAOs, l ‐glutamate oxidase from Streptomyces and l ‐phenylalanine oxidase from Pseudomonas strategically position the helical domains to seal off the active site from the external aqueous environment forming a funnel that has been proposed for substrate entry.…”