Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity.

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k(cat)/K(m) ratio and 7-fold increase in relative activity.

Localization of the ActIII actinorhodin polyketide ketoreductase to the cell wall.

Structurally diverse polyketides provide a rich reservoir of bioactive molecules. Actinorhodin, a model aromatic polyketide, is synthesized by minimal type II polyketide synthase and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. With purified antibodies against actinorhodin polyketide synthase alpha subunit (KSalpha) and ketoreductase, we conducted systematic localization experiments of the two proteins in Streptomyces coelicolor subproteomes. The results support the membrane location of KSalpha and cell-wall location of ketoreductase. Considering previous evidence that some other tailoring enzymes of actinorhodin biosynthesis may be located outside the cytoplasm, a picture is emerging of an extensive role for extracellular biochemistry in the synthesis of type II polyketide antibiotic.

CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor.

Ca(2+) was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known. cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5- agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the Ca(2+) concentration in R5- agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca(2+) concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca(2+)-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a Ca(2+) buffer and exerts its regulatory effects by controlling the intracellular Ca(2+) concentration.

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes.

Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs.

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of K(m) and k(cat). Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket.