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Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways. Alvaro de la Fuente, 2002.A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry . S . clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis . (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a ß-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers . (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain . (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain . An assay for holomycin synthetase was developed . This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor . The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin . Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid. Alexey Teplyakov, 2003.The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown . The crystal structure determination of YchF was sought to help with the functional assignment of the protein . The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 Å resolution . The polypeptide chain is folded into three domains . The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases . An 80-residue domain next to it has a pronounced  -helical coiled coil . The C-terminal domain features a six-stranded half-barrel that curves around an
-helix . The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains . The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein . Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP . Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor .
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