Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.
Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Haren, The Netherlands.
G J
GJ
Euverink
G I
GI
Hessels
C
C
Franke
L
L
Dijkhuizen
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D002826:Chorismate Mutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000096:biosynthesis / Q000494:pharmacology; D011487:Protein Conformation; D014443:Tyrosine / Q000096:biosynthesis / Q000494:pharmacology
D014443:Tyrosine; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D002826:Chorismate Mutase