ABSTRACT
Eukaryotic DNA primase initiates the synthesis of all new DNA strands by synthesizing short RNA oligomers on single-stranded DNA. Additionally, primase helps couple replication and repair and is critical for telomere maintenance and, therefore, chromosome stability. In light of the many aspects of DNA metabolism in which primase is involved, understanding the unique features of the mechanism of this enzyme and how it interacts with other proteins will greatly advance our knowledge of DNA replication and repair.
Subject(s)
DNA Primase/chemistry , DNA Primase/physiology , Eukaryotic Cells/enzymology , Amino Acid Sequence , Animals , DNA Polymerase I/metabolism , DNA Repair/physiology , Humans , Molecular Sequence DataABSTRACT
The carboxy-terminal thioesterase domain of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase catalyzes the hydrolytic release of the tripeptide product (LLD-ACV). By site-directed mutagenesis an S3599A change was introduced into the highly conserved GXSXG motif, resulting in a more than 95 % decrease of penicillin production. Purification of the modified multienzyme showed surprisingly only a 50 % reduction of the peptide formation rate, with the stereoisomer delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine (LLL-ACV) as the dominating product. Thioesterases of ACV synthetases differ from other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXSXG-seryl residue is apparently not essential in acyl transfer leading to peptide release. Instead, this motif may be involved in the control of tripeptide epimerization by selection of the isomer to be released, and the construct supports the presence of LLL-ACV as an intermediate in penicillin biosynthesis.
Subject(s)
Aspergillus nidulans/enzymology , Mutagenesis, Site-Directed/genetics , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/metabolism , Acylation , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution/genetics , Aspergillus nidulans/genetics , Base Sequence , Chromatography, High Pressure Liquid , Conserved Sequence/genetics , Fermentation , Hydrolysis , Kinetics , Oligopeptides/chemistry , Oligopeptides/metabolism , Penicillins/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Synthases/genetics , Peptide Synthases/isolation & purification , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine/genetics , Stereoisomerism , Thiolester Hydrolases/genetics , Thiolester Hydrolases/isolation & purificationABSTRACT
Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.
Subject(s)
Cross-Linking Reagents/chemistry , DNA Primase/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Base Sequence , DNA Primase/chemistry , DNA-Binding Proteins/chemistry , Humans , Kinetics , Photochemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) is synthesised by the multifunctional enzyme ACV synthetase integrating four steps of the penicillin and cephalosporin biosynthetic pathway. Peptide synthesis follows the thiotemplate mechanism from intermediates bound as thioesters to the enzyme. The formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-thioester in the absence of L-valine was shown by isolation of the enzyme-substrate complex and cleavage of the covalently bound intermediate with performic acid. The dipeptide was recovered as cysteic acid or cysteic acid oxime and detected by HPLC and MALDI-TOF mass spectrometry. We conclude that the first peptide bond is formed between delta-carboxyl of L-aminoadipic acid and L-cysteine, followed by addition of the dipeptidyl intermediate to L-valine.
