ABSTRACT
The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.
Subject(s)
Phosphoric Monoester Hydrolases/genetics , Puromycin/biosynthesis , Streptomyces/genetics , Cloning, Molecular , Escherichia coli/genetics , Kinetics , Multigene Family , Nucleotides/metabolism , Streptomyces/enzymology , Streptomyces lividans , Substrate SpecificityABSTRACT
The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.
Subject(s)
Genes, Bacterial , Multigene Family , Puromycin/biosynthesis , Streptomyces/genetics , Streptomyces/metabolism , Chemistry, Physical/methods , Escherichia coli/genetics , Gene Deletion , Gene Expression , Kinetics , Molecular Structure , Plasmids , Puromycin Aminonucleoside/chemistry , Puromycin Aminonucleoside/metabolism , Streptomyces lividans/geneticsABSTRACT
The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.
Subject(s)
Adenosine Kinase/genetics , Adenosine/metabolism , Escherichia coli/enzymology , Saccharomyces cerevisiae/enzymology , Adenosine Kinase/biosynthesis , Adenosine Kinase/isolation & purification , Adenosine Kinase/metabolism , Blotting, Western , Chromatography, Affinity , Deoxyadenosines/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Kinetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
A putative glucose repressor MIG1-homologue (SoMIG1) was isolated from the amylolytic yeast Schwanniomyces occidentalis. Degenerate primers were designed from the conserved zinc finger regions of Mig1 and CreA proteins from different organisms. PCR using these primers and S. occidentalis genomic DNA as template yielded a single 128 bp product. This fragment was used as a DNA probe to screen a S. occidentalis genomic library. Analysis of the positive clones led to the isolation by PCR of a DNA fragment, which contained an open reading frame (ORF) that would encode a 458 amino acid polypeptide. The DNA binding and effector domains of this putative protein showed an identity of 71% and 15%, respectively, to those of the Mig1 protein from Saccharomyces cerevisiae. The SoMIG1 gene complemented a mig1 mutant of this yeast, which suggests that in S. occidentalis SoMIG1 is a glucose repressor. The Accession No. is AJ417892.
