Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase

Abstract

In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubi4, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubi4. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubi4. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychorismate -. 4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.Chorismate is the branch point precursor for the synthesis of many aromatic compounds in Escherichia coli. The seven major end products of chorismate anabolism are phenylalanine, tyrosine, tryptophan, ubiquinone, 4-aminobenzoate, menaquinone, and enterobactin. Chorismate itself undergoes five different conversions that result in those seven products. The tyrosine and phenylalanine pathways diverge following the isomerization of chorismate to prephenate, but two distinct, homologous chorismate mutase activities channel chorismate toward either phenylalanine or tyrosine (10). The menaquinone and enterobactin pathways diverge following the conversion of chorismate to 2-hydroxy-4-deoxychorismate (isochorismate) by isochorismate synthase (28,30). For 4-aminobenzoate synthesis, chorismate is first aminated to form 4-amino-4-deoxychorismate, which then undergoes ,-elimination of the enol-pyruvyl moiety to form 4-aminobenzoate (1,8,21,29). The first step in ubiquinone biosynthesis is the conversion of chorismate to 4-hydroxybenzoate by chorismate lyase (5, 15), in a reaction apparently similar to the second step of 4-aminobenzoate synthesis, catalyzed by 4-amino-4-deoxychorismate lyase. Finally, the first step in tryptophan synthesis is chorismate amination and p-elimination of the enol-pyruvyl group to form anthranilate, or 2-aminobenzoate (2).With the exception of the genes involved in ubiquinone biosynthesis, all of the E. coli genes encoding enzymes that use chorismate or 4-amino-4-deoxychorismate as substrates have been cloned and sequenced. These include pheA (chorismate mutase-prephenate dehydratase), tyrA (chorismate mutase-prephenate dehydrogenase) (10), entC (isochorismate synth...

Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase

“…We determined the K , value of CPL for chorismate as 6.1 pM, which is in reasonable agreement with the previously published value of 9.7 pM (Nichols & Green, 1992). The latter value was obtained with 30 min incubations, which in our hands proved to be beyond time-linearity of the reaction, probably due to the strong product inhibition by PHB which we have observed.…”

“…On the other hand, Nichols & Green (1992) presented evidence which indicates that CPL can convert 4-amino-4-deoxychorismate to 4-aminobenzoate in vim. Therefore a hydroxy or amino function in 4-position appears to be essential for the reaction.…”

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis

“…1 ) ( 21 ). UbiC carries out the fi rst committed step in the biosynthesis of Q 8 , the conversion of chorismate to 4HB ( 22 ). UbiA adds the octaprenyl tail to the 4HB ring, followed by the decarboxylation catalyzed by UbiD and UbiX.…”

Genetic Engineering of Plant Secondary Metabolism (Accumulation of 4-Hydroxybenzoate Glucosides as a Result of the Expression of the Bacterial ubiC Gene in Tobacco)

“…lb). The cloning of ubiC, the gene encoding CPL, was recently reported by our group (Siebert et al, 1992(Siebert et al, , 1994 and by Nichols and Green (1992). The protein was overexpressed, purified, characterized, and shown to be a soluble protein of 19 kD.…”