Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Y Fujishima; P Nordlund; G Pelosi; C J Schofield; S J Cole; J E Baldwin; J Hajdu

doi:10.1006/jmbi.1994.1622

Home/Archive/PubMed

PubMed · 7932729

Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Y Fujishima·P Nordlund·G Pelosi·C J Schofield·S J Cole·J E Baldwin·J Hajdu

View original source ↗Export metadata ↓

Abstract

Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystal forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

Explore related subjects

Acremonium Crystallization Crystallography, X-Ray Escherichia coli Oxidoreductases Recombinant Proteins Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Y Fujishima, P Nordlund, G Pelosi, C J Schofield, S J Cole, J E Baldwin, J Hajdu. 1994-10-07. Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.. https://doi.org/10.1006/jmbi.1994.1622

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs.

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.

Acremonium↗

PubMed2007

Lethal double infection with Acremonium strictum and Aspergillus fumigatus during induction chemotherapy in a child with ALL.

Fungal infections are a major cause of morbidity and mortality in patients during chemotherapeutic treatments and malignant hematologic disease. We present a case of a double fungal infection with disseminated Acremonium strictum (A. strictum) and pulmonary Aspergillus fumigatus (A. fumigatus) and its rapid clinical course. A 17-year-old boy with prolonged neutropenia developed a disseminated fungal infection during induction chemotherapy of his acute lymphoblastic leukemia. The infection was rapidly lethal despite neutrophil recovery and early antifungal combination therapy with amphotericin B and caspofungin. Since there are only a few reports about invasive Acremonium infections, we present this case with regard to differences in the clinic pathologic features of Aspergillosis and other opportunistic fungal infections due to Fusarium or Acremonium species.

Acremonium↗

PubMed2006

An efficient fungal RNA-silencing system using the DsRed reporter gene.

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

Acremonium↗