THE ROLE OF A BETA-BARREL LOOP-4 EXTENSION IN MODULATING THE PHYSICALAND 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 s equencing 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 flavocyto chrome b(2) and glycolate oxidase, the corresponding region of loop is disordered. We now report on the constitutive high-level expression o f isozymes beta(1) and beta(2) in Escherichia coli under control of th e lambda pL promoter, and on the influence of the E. coli genetic back ground and the growth medium on the expression level. We describe the properties of isozyme beta(2) and compare them with those of pure isof orm B-1. The visible spectra of the purified enzymes differ in the pos ition of the near-ultraviolet band of the prosthetic group. pH titrati on 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 isozyme s. 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 spect ra 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 N 3-H bond. Kinetic parameters for a number of substrates and inhibitors are indistinguishable within the limits of experimental error, with t he exception of values for k(cat) for mandelate (the most active subst rate), K-m for hydroxyhippurate (a new substrate), K-i for cinnamate a nd oxalate, and K-d for sulfite. The differences are no larger than tw ofold. The foregoing comparison between isozymes beta(1) and beta(2) s hows that the naturally engineered insertion in loop 4 exerts some inf luence on the flavin spectral properties and the active-site reactivit y. Since the corresponding loop 4 regions in the three-dimensional str uctures of flavocytochrome b(2) and glycolate oxidase are 1.5-2.0 nm r emoved from the flavin, it would appear either that loop 4 has a very different conformation in hydroxy-acid oxidase, or that it may interac t with the active site due to mobility.