A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability.

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

Origin and direction of replication in mitochondrial plasmid DNAs of broad bean, Vicia faba.

Broad bean (Vicia faba) mitochondrial DNA (mtDNA) includes three circular plasmids: mt-plasmid 1 (1,704 ntp), mt-plasmid 2 (1,695 ntp) and mt-plasmid 3 (1,476 ntp). Partially replicated circular forms of these mt-plasmids have been observed in electron microscope preparations. Restriction enzymes that cleave either mt-plasmid 2 (but not mt-plasmids 1 and 3) or mt-plasmid 3 (but not mt-plasmids 1 and 2) were used to generate linear forms of partially replicated mt-plasmid 2 and mt-plasmid 3 molecules. Analyses of these linearized replicative intermediates, observed by electron microscopy, indicated that in both mt-plasmid 2 and mt-plasmid 3 replication originates at a specific location and proceeds in the same, single direction around the molecules. The replication origins of mt-plasmid 2 and mt-plasmid 3 map close to sequences that can fold into hairpin structures.

Ribosomal protein S14 genes in broad bean mitochondrial DNA.

Broad bean (Vicia faba) mtDNA contains an open reading frame with a predicted amino acid sequence that is 41% homologous to the ribosomal protein S14 (RPS14) of Escherichia coli, and which is located 1232 ntp upstream from a gene for cytochrome b (cob). A second putative rpS14 gene occurs in broad bean mtDNA, 344 ntp upstream from a gene for ATPase subunit 9 (atp9). However, the atp9-linked rpS14 gene is 12 codons shorter than the cob-linked rpS14 gene. Sequence homology is found upstream (for 218 ntp) but not downstream from the two rpS14 genes. Transcripts were detected in broad bean mtRNA only for the cob-linked rpS14 gene. All RNA molecules that include a transcript of the rpS14 gene also include a transcript of the cob gene. Sequences homologous to the broad bean mitochondrial rpS14 gene were detected in soybean mtDNA, but not in corn mtDNA. Relationships between the amino acid sequences of RPS14s encoded in broad bean mtDNA, in chloroplast DNAs of various angiosperms, and in E. coli are consistent with the view that the ancestral lines of these three kinds of DNA diverged from each other within a relatively short time period.

Mitochondrial plasmid DNAs of broad bean: nucleotide sequences, complex secondary structures, and transcription.

Three circular plasmid DNA molecules of 1704, 1695 and 1476 nucleotide pairs from broad bean mitochondria (mt-plasmids 1-3) have been sequenced. Within a highly homologous segment of mt-plasmid 1 and 2 are found a series of six directly repeated, inverted repeat sequences, separated by unique sequences. Mt-plasmid 3 contains a series of four inverted repeat sequences, unrelated to the inverted repeat sequences of mt-plasmids 1 and 2. Two RNA molecules of about 440 and 320 nucleotides that are complementary to mt-plasmid 2 were detected. Mapping of 5' and 3' termini of these complementary RNA molecules indicated that all transcription from mt-plasmid 2 occurs within a 441 nucleotide region of the molecule. Evidence for transcription of mt-plasmids 1 and 3 was not found.

A gene for cytochrome c oxidase subunit III (COXIII) in broad bean mitochondrial DNA: structural features and sequence evolution.

A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains the coxIII gene is presented, and compared to corresponding sequences of Oenothera and corn mtDNAs. Upstream from the broad bean coxIII gene are three potential secondary structures: a single stem and loop (hairpin) that is conserved in the Oenothera and corn sequences; a second single stem and loop; and a double stem and loop. The rate of evolution of the coxIII gene has been slower in plants than in mammals. Constraints on the fixation of at least some kinds of mutations in silent (synonymous) third position nucleotides, as well as of mutations that cause amino acid replacements, seem to have contributed to this slower rate.

Sequence and arrangement of the genes for cytochrome b, URF1, URF4L, URF4, URF5, URF6 and five tRNAs in Drosophila mitochondrial DNA.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba has been determined, within which have been identified the 3' end of the large rRNA gene and the entire genes for tRNAleuCUN , URF1 , tRNAserUCN , cytochrome b, URF6 , tRNApro, tRNAthr , URF4L , URF4 , tRNAhis and URF5 . The genes are arranged in the order given, with the large rRNA gene being closest to the A+T-rich region which contains the origin of replication. Transcription of all of these genes except those for cytochrome b, URF6 , tRNAserUCN and tRNAthr proceeds in the same direction as replication. Differences occur in the relative arrangement and in the direction of transcription of these twelve genes between D. yakuba and mammalian mtDNA molecules. Internal AGA codons occur in all of the polypeptide genes except URF6 . Comparisons of the positions of these AGA codons to codons in corresponding mouse genes is consistent with the view that in the D. yakuba mitochondrial genetic code AGA specifies serine. Genes equivalent to all of the polypeptide, tRNA and rRNA genes found in mammalian mtDNA have now been identified in D. yakuba mtDNA.

Expression and assembly of spectrally active recombinant holophytochrome.

To develop an in vitro phytochrome assembly system, we have expressed an oat phytochrome cDNA in both the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. Analysis of soluble protein extracts showed that the recombinant apophytochromes were full-length and capable of covalently attaching the phytochrome chromophore analogue phycocyanobilin. Difference spectra indicated that in vitro-assembled holophytochrome species were photoreversible; however, maxima and minima difference absorption values were blue-shifted relative to those of the native photoreceptor. Extracts containing the recombinant apophytochromes were also incubated with phytochromobilin, the natural chromophore synthesized from biliverdin by cucumber etioplast preparations. In these experiments, the difference spectrum obtained was identical to that of native oat holophytochrome. These results suggest that the recombinant apophytochromes adopt a structure similar to that of the apoprotein biosynthesized in vivo. ELISAs were used to quantitate phytochrome expression levels in both yeast and E. coli extracts. These measurements show that 62-75% of the phytochrome apoprotein in the soluble protein extract was competent to assemble with bilins to form spectrally active holophytochrome.

A broad bean mitochondrial atp6 gene with an unusually simple, non-conserved 5' region.

A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains an atp6 gene of 876 ntp is presented. Relative to other plant atp6 genes, this broad bean gene comprises a 90 ntp non-conserved 5' region, a 759 ntp highly conserved central region and a 27 ntp non-conserved 3' region. The non-conserved, 5' region of the broad bean atp6 gene differs from the corresponding regions of most other plant atp6 genes in that it contains only one potential translation initiation codon and, following this codon, a 63 ntp segment that predicts an amino acid sequence with a predominance of alternating leucines.

A sequence encoding a maturase-related protein in a group II intron of a plant mitochondrial nad1 gene.

We have determined from nucleotide sequence analysis that the subterminal and terminal exons of a respiratory chain NADH dehydrogenase subunit I gene in broad bean mitochondrial DNA (mtDNA) are separated by a group II intron. Within this intron is a 687-codon open reading frame that, from considerations of similarity between amino acid sequences predicted from this open reading frame and maturase-coding sequences in group II introns of certain fungal mitochondrial genes, appears to encode a maturase-related protein. Transcripts complementary to this broad bean sequence (designated a mat-r gene) were detected among RNAs isolated from broad bean mitochondria. Data obtained from DNA-DNA hybridizations indicated that soybean and corn mtDNAs also contain a mat-r gene and suggested that only one copy of this gene occurs in each plant mtDNA. The putative protein specified by the broad bean mat-r gene contains amino acid sequences characteristic of reverse transcriptases. Because of this, consideration is given to the possibility that the maturase-related protein may be functional in the mechanisms by which plant mtDNA sequences are rearranged and foreign sequences are incorporated into plant mtDNAs.

Transfer RNA genes in Drosophila mitochondrial DNA: related 5' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.

Size distributions of circular molecules in plant mitochondrial DNAs.

Some physicochemical properties of the mitochondrial DNAs (mtDNA) from plants of flax, broad bean and mung bean, and from tissue culture cells of jimson weed, soybean, petunia and tobacco were determined. Circular molecules were observed in electron microscope preparations of each mtDNA. In soybean, petunia, broad bean and mung bean mtDNAs, the circular molecules had a continuous distribution of lengths (ranges between 1 to 36 kb, and 1 to 126 kb), heavily skewed toward smaller molecules. Eighty-six percent of the flax circular molecules were from 27 to 54 kb in size, and 78% of the jimson weed circular molecules were from 4 to 15 kb. Replicative forms of 1.2-1.6 kb circular molecules were observed in electron microscope preparations of broad bean mtDNA.

Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum has been determined. This molecule lacks genes for tRNAs of the standard form. Instead, 21 sequences are found that can be folded into structures that resemble tRNAs in which the T psi C arm and variable loop are missing and replaced with a single loop of between 4 and 12 nucleotides. Considerations of various properties of these sequences, including the number, predicted anticodons, conserved nucleotides, direction of transcription, base composition, and relative gene arrangements are consistent with the interpretation that they are genes for a different sort of tRNA. Transfer RNA genes with a similar potential secondary structure are found in mtDNA of the free-living nematode Caenorhabditis elegans, suggesting that this unusual form of tRNA is used by all nematode mitochondria.

Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.

The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile.

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.