Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus.
Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far. An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570. The enzyme has a monomer size of 62 kDa and is processed in the culture to a 33 kDa protein as shown by immunoblotting. The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration. The pure enzyme uses a variety of organic phosphorylated compounds as substrates. The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP. The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S. griseus AP. phoA encodes a protein of 62678 Da with low homology to the AP of Escherichia coli. The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome. On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S. griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.
Moura
Rute S
RS
Instituto de Biotecnologı́a de León INBIOTEC, Parque Cientı́fico de León, Avda del Real no. 1, 24006 León, Spain1.
Martı N
Juan F
JF
Area de Microbioloı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain2.|Instituto de Biotecnologı́a de León INBIOTEC, Parque Cientı́fico de León, Avda del Real no. 1, 24006 León, Spain1.
Martı N
Alicia
A
Area de Microbioloı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain2.
Liras
Paloma
P
Area de Microbioloı́a, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain2.|Instituto de Biotecnologı́a de León INBIOTEC, Parque Cientı́fico de León, Avda del Real no. 1, 24006 León, Spain1.
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase