Mei M. Whittaker

Crystal Structure of Manganese Catalase from Lactobacillus plantarum

BACKGROUND Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.

Glyoxal Oxidase from Phanerochaete chrysosporium Is a New Radical-Copper Oxidase

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl π-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporiumas an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues.

Ligand interactions with galactose oxidase: mechanistic insights

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.

Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.

The Effect of Piericidin A on Energy-Linked Processes in Submitochondrial Particles

Superoxide dismutase (SOD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (a facultative aerobe) has been cloned and expressed in a mesophilic host (Escherichia coli) as a soluble tetrameric apoprotein. The purified apoprotein can be reconstituted with either Mn or Fe by heating the protein with the appropriate metal salt at an elevated temperature (95 °C). Both Mn- and Fe-reconstituted P. aerophilum SOD exhibit superoxide dismutase activity, with the Mn-containing enzyme having the higher activity. P. aerophilum SOD is extremely thermostable and the reconstitution with Mn(II) can be performed in an autoclave (122 °C, 18 psi). The Mn(III) optical absorption spectrum of Mn-reconstituted P. aerophilum SOD is distinct from that of most other MnSODs and is unchanged upon addition of NaN3. The optical absorption spectrum of Fe-reconstituted P. aerophilum SOD is typical of Fe-substituted MnSODs and authentic Fe- SOD and exhibits a pH-dependent transition with an effective pKa value higher than that found for Fe-substituted MnSOD from either E. coli or Thermus spp. Amino acid sequence analysis shows that the P. aerophilum SOD is closely related to SODs from other hyperthermophilic archaea (Aeropyrum pernix and Sulfolobus spp.), forming a family of enzymes distinct from the hyperthermophilic bacterial SOD from Aquifex pyrophilus and from mesophilic SODs.

A Glutamate Bridge Is Essential for Dimer Stability and Metal Selectivity in Manganese Superoxide Dismutase

In Escherichia coli manganese superoxide dismutase (MnSOD), the absolutely conserved Glu170 of one monomer is hydrogen-bonded to the Mn ligand His171 of the other monomer, forming a double bridge at the dimer interface. Point mutation of Glu170 → Ala destabilizes the dimer structure, and the mutant protein occurs as a mixture of dimer and monomer species. The purified E170A MnSOD contains exclusively Fe and is devoid of superoxide dismutase activity. E170A Fe2-MnSOD closely resembles authentic FeSOD in terms of spectroscopic properties, anion interactions and pH titration behavior. Reconstitution of E170A Fe2-MnSOD with Mn(II) salts does not restore superoxide dismutase activity despite the spectroscopic similarity between E170A Mn2-MnSOD and wild type Mn2-MnSOD. Growth of sodA+ and sodA− E. coli containing the mutant plasmid pDT1–5(E170A) is impaired, suggesting that expression of mutant protein is toxic to the host cells.

Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase IMPLICATIONS FOR THE CATALYTIC MECHANISM

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O2-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.

Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris.

The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.

Spectroscopic and magnetochemical studies on the active site copper complex in galactose oxidase

Abstract Galactose oxidase is a radical copper oxidase, an enzyme making use of a covalently modified tyrosine residue as a free radical redox cofactor in alcohol oxidation catalysis. We report here a combination of spectroscopic and magnetochemical studies developing insight into the interactions between the active site Cu(II) and two distinct tyrosine ligands in the biological complex. One of the tyrosine ligands (Y495) is coordinated to the Cu(II) metal center as a phenolate in the resting enzyme and serves as a general base to abstract a proton from the coordinated substrate, thus activating it for oxidation. The structure of the resting enzyme is temperature-dependent as a consequence of an internal proton equilibrium associated with this tyrosine that mimics this catalytic proton transfer step. The other tyrosine ligand (Y272) is covalently crosslinked to a cysteine residue forming a tyrosine–cysteine dimer free radical redox site that is required for hydrogen atom abstraction from the activated substrate alkoxide. The presence of the free radical in the oxidized active enzyme results in formation of an EPR-silent Cu(II) complex shown by multifield magnetic saturation experiments to be a diamagnetic singlet arising from antiferromagnetic exchange coupling between the metal and radical spins. A paramagnetic contribution observed at higher temperature may be associated with thermal population of the triplet state, thus permitting an estimate of the magnitude of the isotropic exchange coupling ( J >200 cm −1 , JS 1 · S 2 ) in this complex. Structural correlations and the possible mechanistic significance of metal–radical coupling in the active enzyme are discussed.