Cloning and characterization of a heterologously expressed bifunctional chorismate synthase/flavin reductase from Neurospora crassa.
Activities of all chorismate synthases (CS) so far analyzed are absolutely dependent upon reduced flavin. For monofunctional CSs, which represent the only class of CSs that have yet been cloned, the flavin must be reduced either (photo-)chemically or by a separable flavin reductase (FR) for in vitro activity. Neurospora crassa CS, in contrast, possesses an intrinsic FR activity and represents the only firmly established member of a bifunctional class of CSs. To better understand this bifunctional protein, a cDNA from an N. crassa expression library encoding a 46.4-kDa protein was cloned by complementation of the CS-deficient Escherichia coli strain AB2849. The deduced amino acid sequence was highly similar (79%) to a previously isolated Saccharomyces cerevisiae CS. The N. crassa sequence was unequivocally shown to encode the bifunctional CS/FR by analysis of the purified protein expressed in E. coli. Based on sequence comparisons with known monofunctional CSs, two regions of 18 internal residues and 29 C-terminal residues unique to N. crassa CS were deleted, and the constructs were also expressed in E. coli. The presence of these regions was found not essential for complementation of the CS- phenotype of E. coli strain AB2849. Although a 3.5-fold decline in specific activity of the purified CS from cells expressing the C-terminal deletion construct was observed, bifunctional activity was not eliminated. These data strongly suggest that the domain(s) responsible for reduction of flavin lie(s) within regions in which homology is also shared among monofunctional CSs.
Henstrand
J M
JM
Institute of Plant Sciences, ETH-Zürich, Switzerland.
Amrhein
N
N
Schmid
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D038181:FMN Reductase; D015870:Gene Expression; D015723:Gene Library; D005800:Genes, Fungal; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D019761:Phosphorus-Oxygen Lyases; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; C095417:chorismate synthase-flavin reductase, Neurospora crassa; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D008190:Lyases; D019761:Phosphorus-Oxygen Lyases