An acid phosphatase from Aspergillus ficuum has homology to Penicillium chrysogenum PhoA.

Three secreted acid phosphatases had previously been characterized from Aspergillus ficuum grown under conditions of limited phosphate. One of these could not be readily separated from AFPhyB, a pH 2.5 optimum acid phosphatase with phytase activity. From extensive protein sequence analysis and subsequent cloning of the gene, we have shown that the AFPhyB protein fraction contains a fourth secreted acid phosphatase (AFPhoA) that has 64% homology to a phosphate-repressible acid phosphatase from Penicillium chrysogenum. Garnier plot analysis revealed that the putative phosphate catalytic domain of AFPhoA at His215Asp216 is similar to those of other acid phosphatases, but that AFPhoA lacks the phosphate-binding motif RHGXRXP of known histidine phosphatases.

The complete primary structure elucidation of Aspergillus ficuum (niger), pH 6.0, optimum acid phosphatase by Edman degradation.

The primary structure of the Aspergillus ficuum (niger) NRRL 3135 extracellular, pH 6.0, optimum acid phosphatase (E.C.3.1.3.2) was elucidated by gas phase sequencing. It was deduced by sequence overlap of peptides obtained from trypsin, chymotrypsin, clostripain, and cyanogen bromide digests of the pyridylethylated protein. The mature, active protein is composed of 583 amino acids, including 13 glycosylated Asn residues. The unglycosylated protein has a MW of 64,245-KDa and a pI of 4.97. Two putative metal binding sites were identified in the molecule. This enzyme may represent a special class of high molecular weight acid phosphatase, since it lacks the active site sequence RHGXRXP and shows no significant homology with known acid phosphatases containing this active site. Homology to human type 5 and A.niger APases was detected, however.

Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum).

An Aspergillus niger (ficuum) genomic DNA lambda EMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase. A clone containing a 1605 bp segment (phyB) encoding the 479 amino acid enzyme was isolated and found to contain four exons. Global alignment revealed 23.5% homology to Aspergillus niger phytase (PhyA); four regions of extensive homology were identified. Some of these regions may contain catalytic sites for phosphatase function.

Identification of active-site residues in Aspergillus ficuum extracellular pH 2.5 optimum acid phosphatase.

Primary structure elucidation of peptides generated by cyanogen bromide, endoproteinase Glu-C, and clostripain cleavage of an Aspergillus ficuum extracellular pH optimum 2.5 acid phosphatase identified a region which contains the active site of the enzyme. The 23-residue segment contains the fragment RHGXRXP, which is homologous to acid phosphatase from Saccharomyces spp., Aspergillus ficuum, mammals, and bacteria. Homologous or conservative substitutions are observed in the 10-amino acid fragment preceding this region.

Aspergillus ficuum phytase: complete primary structure elucidation by chemical sequencing.

The primary structure of Aspergillus ficuum phytase was deduced from overlaps in peptide sequences. The unglycosylated enzyme is a 441 residue protein with a molecular mass of 48.5-KDa, as calculated from the total covalent structure. The estimated pl of the protein is about 4.76. Of the 19 Asn residues, 9 were found to be glycosylated. The phytase consists of 37% non-polar, 42% polar, 11.5% acidic, and 9.5% basic amino acids. The putative active site of the enzyme containing the sequence RHG is located at the N-terminal region of the molecule and shows homology to the active site of both microbial and mammalian acid phosphatases, and phosphoglycerate mutase.

Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway.

The penultimate step in the aflatoxin biosynthetic pathway of the filamentous fungi Aspergillus flavus and A. parasiticus involves conversion of sterigmatocystin to O-methylsterigmatocystin. An S-adenosylmethionine-dependent methyltransferase that catalyzes this reaction was purified to homogeneity (> 90%) from 78-h-old mycelia of A. parasiticus SRRC 163. Purification of this soluble enzyme was carried out by five soft-gel chromatographic steps: cell debris remover treatment, QMA ACELL chromatography, hydroxylapatite-Ultrogel chromatography, DEAE-Spherodex chromatography, and Octyl Avidgel chromatography, followed by MA7Q high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein peak from this step on silver staining identified a single band of approximately 40 kDa. This purified protein was distinct from the dimeric 168-kDa methyltransferase purified from the same fungal strain under identical growth conditions (D. Bhatnagar, A. H. J. Ullah, and T. E. Cleveland, Prep. Biochem. 18:321-349, 1988). The chromatographic behavior and N-terminal sequence of the 40-kDa enzyme were also distinct from those of the 168-kDa methyltransferase. The molar extinction coefficient of the 40-kDa enzyme at 278 nm was estimated to be 4.7 x 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5).

Cyclohexanedione modification of arginine at the active site of Aspergillus ficuum phytase.

Reaction of Aspergillus ficuum phytase with the arginine specific modifier 1,2-cyclohexanedione causes a rapid loss of activity. The inactivation can be partially reversed by 0.2 M hydroxylamine and exhibits pseudo-first order kinetics. The reaction order and second order rate constant of inactivation were 0.87 and 6.72 M-1 Min-1, respectively. Amino acid analysis of modified phytase indicates that about 7 arginine of the total 19 were modified. While the chymotryptic maps of treated and untreated phytase wer virtually identical, the tryptic maps had 4 peaks of altered mobility. An Arg containing tripeptide was identified in the phytase which is also present in other phosphohydrolases and may represent one of the labile Arg involved in the formation of the active site.

High-performance liquid chromatography separation of nikkomycins X and Z.

A reversed-phase, C-18 HPLC method for separation, with baseline resolution, of the chitin synthase inhibitors nikkomycin X and Z is described. This permits, for the first time, satisfactory identification of nikkomycin X and Z contained in a mixture. The use of 30 mM ammonium formate (pH 4.7) containing the ion-pair agent heptanesulfonic acid (1 mM) was critical for the successful separation of these fungicides.

Concurrent disappearance of N-acylethanolamine glycerophospholipids and phagolysosomes enriched in N-acylethanolamine glycerophospholipids as Dictyostelium discoideum cells aggregate.

As the cellular slime mold, Dictyostelium discoideum, undergoes development, a phospholipid fraction containing 80% N-acylethanolamine glycerophospholipids (NAEGPs) and 20% acylphosphatidylglycerol (APG) disappears during the aggregation stage. In this study, the subcellular distribution of that NAEGP phospholipid fraction and the precise time period of disappearance of the fraction were determined. The content of the NAEGP fraction was determined in aggregating cells at 2-h intervals from the beginning of the developmental phase through 14 h, when the cells were completely aggregated. The NAEGP fraction comprised about 8% of the phospholipids in amoebae just starting the development cycle and about 12% in cells between 2 and 6 h of development; then its level decreased until it could not be detected at 12 and 14 h of development. The mole percentage of the total lipid phosphate in the NAEGP fraction was determined in isolated subcellular organelles. The phagolysosomes were enriched in the NAEGP fraction 1.7-2-fold over the level found in the amoebae and about 8-fold over the level in fractions highly enriched in the plasma membrane, mitochondria or peroxisomes. The content of phagolysosomes was determined by electron microscopy of aggregating cells. The amoebae contained large amounts of phagolysosomes up to 6 h of development, and then they gradually disappeared between 6 and 12 h of development. This combination of quantitative phospholipid analysis, subcellular organelle isolation and electron microscopy has revealed that in D. discoideum amoebae, the phagolysosomes were selectively enriched in the NAEGP fraction and both the NAEGP-enriched phagolysosomes and the NAEGPs disappeared concurrently between 6 and 12 h of development.

Comparison of the hydrolysis of phosphatidylethanolamine and phosphatidyl(N-acyl)ethanolamine in Dictyostelium discoideum amoebae.

In Dictyostelium discoideum, (N-acyl)ethanolamine glycerophospholipids disappear as the amoebae aggregate, whereas the amount of ethanolamine glycerophospholipids remains relatively constant, suggesting that each type of ethanolamine-containing phospholipid might have a separate metabolic pathway. To study their metabolism, phosphatidylethanolamine and phosphatidyl(N-acyl)ethanolamine containing either [14C]ethanolamine or a 14C-labeled sn-2 fatty acyl group were incubated with D. discoideum homogenates, and the conversion of the substrates into radioactive products was monitored. At pH values 3.8 and 4.5, phosphatidyl(N-acyl)ethanolamine was hydrolyzed by a phospholipase A1 to form the sn-2 acyl form of the lipid. Only minor hydrolysis occurred at pH values of 5.2 or higher. (N-acyl)Ethanolamine was also released by a phospholipase D type activity at 0.1 the rate of the lysophospholipid formation. Phosphatidyl(N-acyl)ethanolamine was not hydrolyzed to form phosphatidylethanolamine or water soluble components. At pH 7.2 and at the low pH range of 3.8-4.5, phosphatidylethanolamine was hydrolyzed to lysophosphatidylethanolamine, which was then further degraded to water-soluble components. At pH 7.2, a phospholipase A2 initially hydrolyzed the phosphatidylethanolamine, whereas at the low pH range a phospholipase A1 was the most active enzyme. Although both types of ethanolamine-containing phospholipid were hydrolyzed by a phospholipase A1 at the low pH range, phosphatidylethanolamine hydrolysis was more sensitive to inhibition by Trition X-100, and phosphatidylethanolamine was hydrolyzed to water-soluble components, whereas phosphatidyl(N-acyl)ethanolamine was not. At pH 7.2, phosphatidylethanolamine was hydrolyzed, but phosphatidyl(N-acyl)ethanolamine was not hydrolyzed at all. These results indicate that there are separate routes of degradation for the two types of ethanolamine-containing phospholipid in D. discoideum.