Crystal Structure Analysis of Recombinant Rat Kidney Long Chain Hydroxy Acid Oxidase^,

Abstract

Long chain hydroxy acid oxidase (LCHAO) is a member of an FMN-dependent enzyme family that oxidizes L-2-hydroxy acids to ketoacids. LCHAO is a peroxisomal enzyme, and the identity of its physiological substrate is unclear. Mandelate is the most efficient substrate known and is commonly used in the test tube. LCHAO differs from most family members in that one of the otherwise invariant active site residues is a phenylalanine (Phe23) instead of a tyrosine. We now report the crystal structure of LCHAO. It shows the same beta8alpha8 TIM barrel structure as other structurally characterized family members, e.g., spinach glycolate oxidase (GOX) and the electron transferases yeast flavocytochrome b2 (FCB2) and Pseudomonas putida mandelate dehydrogenase (MDH). Loop 4, which is mobile in other family members, is visible in part. An acetate ion is present in the active site. The flavin interacts with the protein in the same way as in the electron transferases, and not as in GOX, an unexpected observation. An interpretation is proposed to explain this difference between GOX on one hand and FCB2 and LCHAO on the other hand, which had been proposed to arise from the differences between family members in their reactivity with oxygen. A comparison of models of the substrate bound to various published structures suggests that the very different reactivity with mandelate of LCHAO, GOX, FCB2, and MDH cannot be rationalized by a hydride transfer mechanism.

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

“…16 These two compounds were further characterized in a spectrum of assays, including intervention studies in a wellestablished deoxycorticosterone acetate (DOCA) salt hypertension model. Molecular modeling studies using the rat Hao2 crystal structure (Protein Data Bank entry 1TB3) 17 were used to understand the potential binding mode of the inhibitor (3) in the enzyme active site. The modeling suggests that the carboxylic acid moiety binds in the active site by forming salt bridge interactions with basic residues R250 and R164 (Figure 1).…”

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

“…They share a common (b/a) 8 TIM barrel fold, with the FMN binding site located at the C-terminal end of the b barrel [3][4][5][6][7]. Enzymes are functional oligomers, typically homotetramers.…”

“…L-lactate, glycolate, L-mandelate) via C-H hydrogen abstraction [8][9][10][11][12][13][14], probably via hydride transfer to the oxidized form of FMN [15,16]. The residues involved in FMN binding and forming the active site are highly conserved [3][4][5][6][7], suggesting a common catalytic mechanism of substrate oxidation in the first (reductive) half-reaction of the overall conversion ( Fig. 1, Scheme 1).…”

“…Small differences in microenvironment of the reactive C4a-N5 locus of FMN might be responsible [17,18], in part at least, for these starkly varied O 2 reactivities. Enzyme structures [3][4][5][6][7] reveal that strand b1 of the b barrel provides close contact contact with the re-face, the side of the flavin ring opposite to where the substrate binds (Fig. 1).…”

The 2.1 Å structure ofAerococcus viridansL-lactate oxidase (LOX)

“…The imine product is then spontaneously hydrolysed to yield an -hydroxy acid and NH þ 4 (Walsh et al, 1971). The structures of the four related enzymes glycolate oxidase (GLO; PDB code 1gox; Lindqvist & Branden, 1989), a soluble chimeric form of mandelate oxidase (MDH; PDB code 1p4c; Sukumar et al, 2004), long-chain hydroxy-acid oxidase (HAO; PDB code 1tb3; Cunane et al, 2005) and flavocytochrome b 2 (B2; PDB code 1fcb; also known as lactate dehydrogenase; Xia et al, 1987) have been determined by X-ray crystallography. In each of these struc-tures, the monomer contains a classic eight-stranded /-barrel with binding sites for the FMN cofactor and substrate at the C-terminal end of the -barrel.…”