Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination.

A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with 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 manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.

Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis.

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4-alpha-D-galacturonide)galacturonohydrolase] that cleaves monomer units from the non-reducing end of the substrate molecule. K(m) and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml-1 and 3.1 mg ml-1 and 255 U mg-1 and 262 U mg-1 for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2-7), the Michaelis constant (K(m)) decreased with increasing chain length (n). The Vmax value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki = 0.3 mM) as well as by reduced digalacturonate (Ki = 0.4 mM). The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.

Characterization of manganese(II) binding site mutants of manganese peroxidase.

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 manganese binding site of manganese peroxidase: characterization of an Asp179Asn site-directed mutant protein.

A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the Km for the substrate MnII was approximately 50-fold greater than the corresponding Km for the wild-type recombinant enzyme (3.7 mM versus approximately 70 microM). Likewise, the kcat value for MnII oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent Km for H2O2 of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by MnII was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (KD) for MnP D179N compound II reduction by MnII was approximately 100-fold greater than the KD for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of MnII to the enzyme and, consequently, the rate of compound II reduction by MnII. In contrast, the mutation apparently does not have a significant effect on H2O2 cleavage during compound I formation or on p-cresol reduction of compound II.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.

The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans.

Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger.

Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the laboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commercial pectinase preparation Ultrazym. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5' or 3' parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym, PLII.