In vitro biosynthesis of peptolide SDZ 90-215 by a 1.2 MDa multienzyme polypeptide.
An enzyme fraction with catalytic activities for the biosynthesis of the pipecolic acid containing cyclopeptolide SDZ 90-215 was partially purified and characterized from the genus Septoria. The crude cell homogenate was subjected to polyethyleneimine precipitation, ammonium sulfate precipitation and FPLC gel filtration. The denatured enzyme shows an apparent molecular mass of 1.2 MDa in 3% SDS-PAGE. Peptolide SDZ 90-215 synthetase catalyzes the ATP-PPi exchange reaction dependent on all substituent amino and hydroxy acids. SDZ 90-215 synthetase synthesizes the peptolide in vitro when incubated together with D-lactic acid, all constitutive amino acids in their N-unmethylated form, ATP, magnesium chloride and S-adenosyl-L-methionine. The yield of SDZ 90-215 is higher when O-methyl-L-tyrosine instead of L-tyrosine is used, indicating that O-methylation of tyrosine is not carried out by the synthetase.
Lee
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Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Federal Republic of Germany.
Lawen
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D008781:Methyltyrosines / Q000737:chemistry; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010453:Peptide Synthases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010875:Pipecolic Acids / Q000032:analysis; D014443:Tyrosine / Q000378:metabolism
D003470:Culture Media; D008781:Methyltyrosines; D009097:Multienzyme Complexes; D010456:Peptides, Cyclic; D010875:Pipecolic Acids; C086371:SDZ 90-215; C002350:O-methyltyrosine; D014443:Tyrosine; D010453:Peptide Synthases; C086372:SDZ 90-215 synthetase; C031345:pipecolic acid