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K Lerch

Publications and source records attributed to K Lerch.

At least 19 recordsLinked to original sources
PubMed1992

Stabilization of the oxy form of tyrosinase by a single conservative amino acid substitution.

Asp-208 of Streptomyces glaucescens tyrosinase (an invariant residue in the CuB-binding region of tyrosinases and haemocyanins) was conservatively substituted by glutamic acid. Although having little effect on spectroscopic or kinetic properties of the enzyme, the mutation greatly decreased the lability of Cu-bound O2. A rationalization for these results is given, based on the crystal structure of Panuliris interruptus haemocyanin in the conserved CuB-binding region.

Amino Acid Sequence↗
PubMed1992

Expression of Neurospora crassa laccase under the control of the copper-inducible metallothionein-promoter.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. In vegetatively growing cultures its biosynthesis is repressed but can be induced by different protein synthesis inhibitors. Transformation of the N. crassa wild-type strain Singapore with a fusion gene consisting of the N. crassa copper-metallothionein promoter and the laccase gene are described in this report. Correct integration of the 3.6 kilobase (kb) promoter-fragment fused with the laccase gene containing a 5' consensus region leads to copper-dependent expression of the enzyme during the vegetative growth phase. The enzyme is glycosylated and secreted, and high amounts of extracellular activity can be detected. The regulation of laccase biosynthesis of one examined transformant, followed at both the transcriptional and the translational level, indicates co-induction of both copper-metallothionein and laccase. The data presented show that expression of the recombinant laccase gene is exclusively regulated by the transformed N. crassa metallothionein-promoter.

Blotting, Southern↗
PubMed1991

Regulation of biosynthesis of L-amino acid oxidase by Neurospora crassa.

L-Amino acid oxidase was purified from liquid cultures of Neurospora crassa induced by L-phenylalanine, D-phenylalanine, ATP and cycloheximide. Although the four enzyme species isolated were found to differ in size and electrophoretic mobility, they were the product of a single gene as demonstrated by genomic Southern analysis. Northern analysis of total cellular RNA showed a rapid increase of L-amino acid oxidase mRNA in response to the different inducing agents studied. These data suggest that the L-amino acid oxidase gene from N. crassa is regulated at the transcriptional level.

Adenosine Triphosphate↗
PubMed1991

Albino mutants of Streptomyces glaucescens tyrosinase.

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

Albinism↗
PubMed1991

Regulation of laccase synthesis in induced Neurospora crassa cultures.

Rapidly growing cultures of N. crassa do not produce laccase. Exposure of this fungus to different inducing agents leads to a de novo biosynthesis of extracellular laccase in vegetative cultures. In this study the induction of laccase after addition of cycloheximide and D-phenylalanine is reported. De novo synthesis of laccase mRNA was followed over 96 h after induction. A fast appearance of the message, as well as its presence over a rather long period, indicates a regulation on a transcriptional and maybe on a post-transcriptional level. In contrast to the kinetics of mRNA production, Western analysis with a polyclonal anti-laccase antibody showed a remarkably delayed appearance of the intracellular, as well as of the extracellular, protein product after induction with cycloheximide. Furthermore, activity measurements at different times after induction of both crude extracts and media of the vegetative cultures showed that in extracted mycelia the activity occurs at least 20 h after the protein is immunologically detectable. Laccase activity in the medium starts to increase only 30 h after translation. These data, together with the published structure of the laccase gene, indicate a regulation on the transcriptional, post-transcriptional and on a post-translational level. In cultures induced with D-phenylalanine a rather fast appearance of laccase-specific mRNA also indicates a transcriptional regulation. Compared to cycloheximide-induced laccase biosynthesis no delayed appearance of laccase protein levels of laccase activity is observed after induction with D-phenylalanine.

Blotting, Western↗
PubMed1990

Molecular cloning of the L-amino-acid oxidase gene from Neurospora crassa.

The addition of D-phenylalanine to starved cultures of Neurospora crassa leads to de novo synthesis of L-amino-acid oxidase. Poly(A) RNA from D-phenylalanine-treated mycelium was therefore used to generate a cDNA library which was subsequently screened by hybrid-selected translation. A positive L-amino-acid oxidase clone served as a probe to isolate the complete gene from a genomic library of N. crassa. The nucleotide sequence obtained revealed an open reading frame coding for a protein of 695 amino acids. A comparison of the deduced primary structure with the partial amino-terminal sequence of the isolated enzyme showed that the protein is synthesized as a precursor. The proform exceeds the mature enzyme by 129 amino acids. The presence of a cluster of basic amino acid residues preceding Ala129 in the precursor suggests a post-translational modification brought about by limited proteolysis. N. crassa L-amino-acid oxidase shares a highly conserved region with many well-characterized flavoproteins that is known to constitute part of the flavin-adenine dinucleotide-binding site.

Amino Acid Oxidoreductases↗
PubMed1990

The reaction of CN- with the binuclear copper site of Neurospora tyrosinase: its relevance for a comparison between tyrosinase and hemocyanin active sites.

The equilibrium and the kinetics of the reaction between Neurospora crassa tyrosinase and cyanide have been studied. Cyanide reacts with the binuclear copper active site of the protein competitively with respect to dioxygen and displaces the metal ions. This process occurs stepwise and involves transient intermediates containing mononuclear Cu(I) sites. The reaction mechanism proved to be the same as described earlier for molluscan and arthropodan hemocyanins, which share with tyrosinase the same copper active site organization, but perform different physiological functions. A comparison of the kinetic parameters between the different proteins shows that the tyrosinase copper active site has a greater accessibility than that of hemocyanin. The relevance of these data in terms of structure-function relationship and evolution of the binuclear copper proteins is discussed.

Binding Sites↗
PubMed1990

Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene.

Wild-type Neurospora crassa, strain Singapore, was transformed with a N. crassa metallothionein promoter/protyrosinase fusion gene. Transformants produced tyrosinase during vegetative growth, as determined by Western analyses and activity assays. This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. Complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosinase, whereas a 3.6 kb promoter-fragment conferred copper inducibility on the reporter gene in four transformants. A transformant with high constitutive tyrosinase levels was able to produce melanin on complete medium agar plates supplemented with 1 mg/ml L-tyrosine.

Cloning, Molecular↗
PubMed1990

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa.

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.

Adenosine Triphosphate↗
PubMed1989

Isolation and characterization of the tyrosinase gene from Neurospora crassa.

A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.

Amino Acid Sequence↗
PubMed1989

Luminescence emission from Neurospora copper metallothionein. Time-resolved studies.

The luminescence lifetime of Cu-metallothionein from the fungus Neurospora crassa has been studied by the frequency-domain emission technique. Lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. Binding of Hg(II) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. No quenching by oxygen is found for the Hg(II)-Cu-metallothionein adduct. By analogy to model compounds, luminescence emission is attributed to a triplet excited state of a Cu(I)-to-sulphur charge-transfer complex. The comparison of bimolecular quenching constants for O2 and acrylamide indicates a highly compact structure of the protein.

Luminescent Measurements↗
PubMed1989

Proton NMR studies of a metallothionein from Neurospora crassa: sequence-specific assignments by NOE measurements in the rotating frame.

Sequential 1H NMR assignments of a metallothionein from Neurospora crassa have been accomplished by the combined use of COSY, 2QF-COSY, HOHAHA, and rotating-frame NOE experiments. All potentially observable resonances were assigned except for the epsilon-NH3 group of the C-terminal lysine. 1H NOEs, when observed in the laboratory frame and at 500-MHz spectrometer frequency, were negligible in this protein due to the inherent rotational correlation time of the molecule. This difficulty was circumvented by measuring transverse NOEs in the rotating frame under spin-locking conditions. The observed pattern of NOEs reveals a marked absence of "regular" secondary structures in the protein. Thus, the stability of this metallothionein's tertiary structure must arise primarily from its metal ligation. This appears to be a general feature of MTs since a general lack of extensive secondary structural elements was also observed in other metallothioneins.

Amino Acid Sequence↗
PubMed1989

The inhibition of tyrosinase by pyridinones.

3-Hydroxypyridine-4-ones have potential as orally active chelators of iron(III) and therefore may find application in the treatment of thalassaemia. An undesirable feature of these molecules is that they inhibit tyrosinase. We have established that alkyl substitution at position 2 in the aromatic ring minimizes interaction with tyrosinase and does so without appreciably influencing the affinity for iron(III).

Catechol Oxidase↗
PubMed1988

Unprecedented lysyloxidase activity of Pichia pastoris benzylamine oxidase.

Benzylamine oxidase (EC 1.4.3.6) from the yeast Pichia pastoris is a 106 kDa quinoprotein containing one copper atom per molecule. It has a broad substrate specificity ranging from butylamine to peptidyl lysine in collagen and elastin. The kinetic data obtained using lysine-containing model peptides as substrates indicate an astonishing similarity to mammalian lysyloxidase. This similarity is further supported by the inhibition of both enzymes with beta-aminopropionitrile.

Amino Acid Oxidoreductases↗
PubMed1988

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase.

The physiochemical properties of wild type and two mutants of Streptomyces glaucescens tyrosinase are reported. The native enzyme contains two coppers at the active site which are EPR nondetectable. The two coppers react stoichiometrically with one hydrogen peroxide molecule giving rise to oxytyrosinase. Its optical features are similar to those reported earlier for a molluscan hemocyanin. The two mutants in which histidine-62 and -189 were changed to asparagine by site-directed mutagenesis have lost their enzymatic activity and their ability to bind oxygen and contain only one copper ion which is fully EPR detectable. The EPR parameters indicate that the remaining copper is in a tetragonally distorted ligand environment. These data are in agreement with His-62 and His-189 serving as copper ligands in S. glaucescens tyrosinase.

Amino Acid Sequence↗
PubMed1988

Luminescence of deoxyhemocyanin and deoxytyrosinase.

The deoxy form of hemocyanins and tyrosinases from certain species displays a weak low-energy luminescence when solutions of the protein are irradiated with light at approximately 290 nm. The emission most likely results from a copper-to-imidazole charge transfer state as shown by studies with a synthetic copper(I) complex having three imidazole ligands.

Animals↗
PubMed1988

Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor.

The complete structures of the laccase genes isolated from two different Neurospora crassa wild-type strains are described. The genes were cloned by screening partial genomic DNA libraries with a nick-translated laccase-specified 1.36-kilobase SalI fragment (Germann, U. A., and Lerch, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8854-8858) as a hybridization probe. Nucleotide sequence analysis revealed the presence of two different allelic forms. They conform to the same structural organization, but show an overall divergence of 5.3% which is mainly the result of point mutations in the nontranslated regions. The coding parts are interrupted by a short intron. The encoded proteins differ in 12 out of 619 amino acid residues. A comparison of the primary structure deduced from the nucleotide sequence of the gene with a protein chemical analysis of the two terminal cyanogen bromide fragments of extracellular N. crassa laccase revealed that the enzyme is synthesized as a precursor. The precursor protein exceeds the mature protein by 49 amino acids at its amino terminus and by 13 amino acids at its carboxyl terminus, thus indicating a complex maturation pathway. The possible involvement of amino-terminal processing in secretion and of carboxyl-terminal processing in activation of the enzyme is discussed.

Alleles↗