The role of a beta barrel loop 4 extension in modulating the physical and functional properties of long-chain 2-hydroxy-acid oxidase isozymes

Peroxisomal long-chain 2-hydroxy-acid oxidase, an FMN-dependent enzyme, catalyzes the oxidation of a variety of L-2-hydroxy acids into keto acids at the expense of oxygen. We recently reported the cloning and sequencing of its cDNA and the existence of a weakly expressed isozyme [Belmouden, A., Le, K. H. D., Lederer, F. & Garchon, H. J. (1993) Eur: J. Biochem. 214, 17-25]. This isozyme. beta(2) differs from the major one in having a three-residue insertion, -VRK-, in loop 4 of the beta(8) alpha(8) barrel. In the crystal structures of homologous flavocytochrome b(2) and glycolate oxidase, the corresponding region of loop is disordered. We now report on the constitutive high-level expression of isozymes beta(1) and beta(2) in Escherichia coli under control of the lambda pL promoter, and on the influence of the E. coli genetic background and the growth medium on the expression level. We describe the properties of isozyme beta(2) and compare them with those of pure isoform B-1. The visible spectra of the purified enzymes differ in the position of the near-ultraviolet band of the prosthetic group. pH titration studies indicate that the FMN ionizes at N3 at a lower pH than free flavin and that there is a small pK(a) difference between the isozymes. To our knowledge, the only other known case of a lowered pK(a) for the protein-bound flavin is that of glycolate oxidase. In the CD spectra of the FMN region, a marked difference between isozymes in the 170-300-nm region appears to be related to the pK(a) difference for that N3-H bond. Kinetic parameters for a number of substrates and inhibitors are indistinguishable within the limits of experimental error, with the exception of values for k(cat) for mandelate (the most active substrate), K-m for hydroxyhippurate (a new substrate), K-i for cinnamate and oxalate, and K-d for sulfite. The differences are no larger than twofold. The foregoing comparison between isozymes beta(1) and beta(2) shows that the naturally engineered insertion in loop 4 exerts some influence on the flavin spectral properties and the active-site reactivity. Since the corresponding loop 4 regions in the three-dimensional structures of flavocytochrome b(2) and glycolate oxidase are 1.5-2.0 nm removed from the flavin, it would appear either that loop 4 has a very different conformation in hydroxy-acid oxidase, or that it may interact with the active site due to mobility.