Structural studies on a mitochondrial glyoxalase II

Abstract

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

Fig. 1

Sequence alignment of GLX2-5 from A. thaliana and GLX2 from human.

Fig. 2

EPR spectrum of GLX2-5. Trace A shows the spectrum of GLX2-5 recorded at 4.7 K. The inset shows an expanded view of the g ~ 1.9 region of the spectrum (B) and a theoretical spectrum (C). The theoretical spectrum was generated using the spin Hamiltonian H = βg₁.B.S₁ + S₁.D₁.S₁ + βg₂.B.S₂ + S₂.D₂.S₂ + J.S₁.S₂, where S₁ = 5/2, g_1(iso) = 2.0, D₁ = 3 cm⁻¹, E₁ = 0, S₂ = 1, g_2(x,y,z) = 2.093, 2.192, 2.322, D₂ = 10 cm⁻¹, E = 0, and J = 50 cm⁻¹. This scheme represents a strongly antiferromagnetically coupled Fe(III)-Fe(II) center. Trace D shows a composite simulation of the experimental EPR spectrum. The simulation has two components, one of which is that shown as Trace C, and another, the major component, corresponds to an isolated Fe(III) ion with H = βg.B.S + S.D.S, where g_(iso) = 2.0, S = 5/2, D = 0.45 cm⁻¹, E = 0.0855 cm⁻¹ (E/D = 0.19), ΔD = 0.20 cm⁻¹, and ΔE = 0.057 cm⁻¹. An additional component with an isotropic g_(eff) = 4.3 is present but was not included in the simulation.

Fig. 3

NMR spectrum of 1.6 mM GLX2-5. NMR spectra were collected on a Bruker Avance 500 spectrometer operating at 500.13 MHz, 298 K, and a magnetic field of 11.7 T, recycle delay (AQ), 41 ms; sweep width, 400 ppm. Protein chemical shifts were calibrated by assigning the H₂O signal the value of 4.70 ppm. A modified presaturation pulse sequence (zgpr) was used to suppress the proton signals originating from water molecules.

Fig. 4

Ribbon structure of GLX2-5 from Arabidopsis thaliana. The coordinates have been deposited on the Protein Databank (accession number: 1XM8). Figure was rendered using Raswin Molecular Graphics, Windows version 2.7.2.1.1.

Fig. 5

Active site of GLX2-5 from Arabidopsis thaliana. The coordinates have been deposited on the Protein Databank (accession number: 1XM8). Figure was rendered using Raswin Molecular Graphics, Windows version 2.7.2.1.1.

Fig. 6

Elution profile from gel filtration column. Approximately 5 mg of GLX2-5, 7 mg Ovalbumin, 10 mg Ribonuclease A, and 1 mg Blue Dextran were separated on a Sephadex S200 column. Samples containing protein were identified by monitoring A₂₈₀ and by SDS-PAGE.