Characterization of Manganese(II) Binding Site Mutants of Manganese Peroxidase

Abstract

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

The Role of Glu39 in MnII Binding and Oxidation by Manganese Peroxidase from Phanerochaete chrysoporium

“…The D179A and E35D variants exhibited characteristics similar to the previous single variant MnPs (D179N and E35Q), including increased K m for Mn II and decreased k cat values (37). In striking contrast to the previous E39Q variant (35), the E39D variant was claimed to exhibit wild-type characteristics including wildtype kinetics for Mn II binding and oxidation under both steady-state and transient-state conditions (37). Those investigators concluded that E39 was "not critically important" to Mn II binding nor electron transfer from Mn II to the enzyme.…”

“…Site-directed mutations were introduced into the pGM1 plasmid (35), which contains 1.1 kb of the gpd promoter fused to the coding region of P. chrysosporium mnp1 at the ATG translation initiation codon, by the PCR-based Quikchange (Stratagene) method. Forward and reverse primers (16)(17)(18)(19)(20) bp) containing altered codons were obtained to introduce the mutations for each amino acid.…”

“…These stationary cultures were homogenized and used as inocula for shaking cultures containing 1 L of liquid medium in 2-L flasks, and were grown for 3 days at 28°C. The extracellular medium was filtered and concentrated, and the variant MnP proteins were purified by a combination of Phenyl Sepharose CL-6B hydrophobic interaction, Cibacron blue 3GA dye affinity, and MonoQ anionic exchange chromatographies as described previously (35,42 kinetic experiments were performed using an Applied PhotoPhysics SX.18MV sequential stopped-flow reaction analyzer at 25.0 ( 0.2°C as described (45). Reductions of each intermediate by ferrocyanide were measured individually.…”

“…Extracellular medium from the transformants was tested for MnP activity (15), and the transformant exhibiting the highest activity was selected for further study. Enzymes were purified from the extracellular medium by successive Phenyl Sepharose, Blue Agarose affinity, and MonoQ anion exchange chromatographies (35,42). The yields were comparable to those for previous variants and recombinant wild-type MnP (∼2 mg‚L -1 ) (34,35,42,45).…”

High-Resolution Crystal Structure of Manganese Peroxidase: Substrate and Inhibitor Complexes,

“…Alteration of the proposed ligands in the Mn-binding site significantly affects Mn binding and oxidation (17)(18)(19)(20)(21)(22), and crystals of both the single variant, D179N, and the double variant, E35Q/D179N, lack electron density at the proposed Mn-binding site (23), suggesting that Mn II is not bound. MnP is unique among enzymes using manganese as a redox cofactor.…”