Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum.

The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (delta psi) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H+ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H+-arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.     

Uptake of phenoxyacetic acid by Penicillium chrysogenum

The uptake of phenoxyacetic acid by two different strains of Penicillium chrysogenum was studied. Phenoxyacetic acid (POA) was taken up by P. chrysogenum in a defined medium. Plots of initial velocity of POA uptake versus external substrate concentration, in the range 2–5000 M, gave linear plots. Uptake of POA by induced and uninduced cells was identical. The initial velocity of POA uptake decreased as the pH of the suspension was increased from 5.4 to 7.2; the decrease closely paralleled the decline in the non-ionic form of the acid over this pH range. The initial velocity of POA uptake was not affected by the presence of phenylacetic acid. POA uptake proceeded until the cellular concentration was equal to the external concentration. It is concluded that POA is passively transported into P. chrysogenum by unmediated diffusion.     

Guanyl-specific ribonuclease from the fungus Penicillium chrysogenum strain 152 and its complex with guanosine 3'-phosphate studied by nuclear magnetic resonance

The method of proton magnetic resonance was used to obtain information on the active site of the guanyl-specific ribonuclease from Penicillium chrysogenum, strain 152A. Four pH-dependent signals in the aromatic region of the proton NMR spectrum of the enzyme were assigned to the C-2 and C-4 protons of the two histidine residues. To determine the pK values and the environment of the histidine residues the pH dependence of their chemical shifts was studied and experimental curves thus obtained were analyzed taking into account the effect of other dissociating groups of the enzyme. The pK values of the histidine residues were found to be equal to 7.92 +/- 0.04 and 7.86 +/- 0.09. The results of the calculations indicate that each histidine residue should interact with an acidic group (carboxylic) of the protein (pK 4.33 and 3.48) and the distance between two histidine residues does not exceed 0.85 nm. The rate constants for the quasi-first order reaction of deuterium exchange of the histidine residues (11.2 s-1 and 3.7 x-1) suggest that both residues are accessible, though to a different degree to solvent. Formation of a complex between the enzyme and guanosine 3'-phosphate (Guo3'P) is accompanied by the shift of the histidine pK toward the alkaline region by 0.5. The existence of the complex is controlled by dissociation of a histidine residue with pK 8.7 in alkaline medium and by protonation of the N-7 of Guo3'P (pK 2.4) in acid medium. Nuclear Overhauser effect measurements were used to determine the glycosidic torsion angle for the Guo3'P in the complex and to estimate the distances between the histidine residues of the enzyme and ribose ring of Guo-3'P. The results obtained suggest that the nucleotide in the complex has an anti conformation and the least exposed histidine is spaced not more than 0.5 nm from the C-1' proton of the nucleotide ribose ring. A model for the enzyme-nucleotide complex is presented.     

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l-leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l-leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase).The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.     

Extracellular guanyl-specific ribonuclease Sa from the actinomycete Streptomyces aureofaciens. Primary structure and homology with ribonucleases from bacteria and fungi

The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.     

Extracellular RNAase Pch2 of Penicillium chrysogenum 152A: purification, specificity and physico-chemical properties

Acid RNAase Pch2 was isolated from a filtrate of the cultural fluid of the fungus Penicillium chrysogenum 152A and purified to homogeneity. An analysis of RNAase Pch2 action on RNA and synthetic substrates showed that the enzyme can be attributed to non-specific true ribonucleases (ribonucleate-3'-oligo-nucleotide hydrolase, EC 3.1.4.23). The maximal effect of the enzyme on RNA is observe at pH 4.5 and 55 degree. The RNAase Pch2 is not activated by bivalent metal ions, p-chloromercurybenzoate or beta-mercaptoethanol and is reversibly inactivated by 8 M urea. The enzyme molecule consists of 332 amino acid residues; its molecular weight is 36160, the isoelectric point lies at 5.2.     

A circular dichroism study of the structure of Penicillium chrysogenum mycovirus

We have examined the absorption and circular dichroism spectra of intact Penicillium chrysogenum virus, empty capsid particles, and isolated double-stranded RNA. The absorbance at 260 nm of intact virus was less than 4% hypochromic relative to the absorbances of the free double-stranded RNA and free viral protein, indicating very little change in the base stacking interactions of the RNA. Circular dichroism studies of intact virus indicate that the capsid protein consists of 45% alpha-helix. Empty capsids, containing a protein of the same molecular weight as intact virus protein, were found to have 30% alpha-helix, suggesting a conformational change in the capsid upon assembly with RNA. The conformation of double-stranded RNA in the virus was slightly altered from the solution structure of the RNA in 0.01 M Na+ and resembled the conformation of double-stranded RNA partially bound with spermidine. However, the virus does not appear to contain polyamines. Electrophoretic experiments indicate a pH- and salt-titratable RNA binding site on the capsid protein in virus disrupted by urea or non-ionic detergents. The results are consistent with significant ionic interactions between the RNA and the capsid protein in the virus.