Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli

Abstract

A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome. Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase. DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine. The deduced amino acid sequence for the 47.2-kilodalton subunit of E. coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N-and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide.When microbes grow on C2 compounds or when fatty seeds germinate, function of the glyoxylate shunt ensures the net conversion of acetyl coenzyme A (acetyl-CoA) and C4 intermediates of the tricarboxylic acid cycle (24). Two enzymes of the shunt, isocitrate lyase and malate synthase, are inducibly synthesized under such metabolic conditions, posing an interesting question about regulation of the glyoxylate shunt at the level of enzyme activity and gene expression. In the most-studied case at the genetic level, the structural genes for malate synthase, aceB, and isocitrate lyase, aceA, are located sequentially at ca. 90 min of the Escherichia coli K-12 linkage map and constitute an operon negatively controlled by a neighboring regulatory gene, iciR (6, 29). In addition, dual control of the ace operon by iciR and fadR, a regulatory gene for fatty acid degradation, has been elucidated (29). The ace operon accommodates another gene, aceK, specifying a kinase/phosphatase that controls isocitrate dehydrogenase via reversible phosphorylative inactivation (8,26,27). Of particular interest is the recent finding that E. coli isocitrate lyase may be regulated by phosphorylative activation (39). Thus, the utilization of isocitrate carbon may be controlled after the branch between the tricarboxylic acid cycle and glyoxylate shunt by the activity of isocitrate dehydrogenase and isocitrate lyase, which are oppositely regulated by phosphorylation.Of additional interest is the localization of the glyoxylate shunt enzymes in plant cellular microbodies termed glyoxysomes, which are a subcategory of peroxisomes (2). The molecular weight of isocitrate lyase, which is a tetrameric protein in most instances so far examined (18,20,21,32,38), has been shown to be approximately 33% higher in plants and fungi than in procaryotes.We now report an analysis of the aceA region of E. coli by gene cloning and the primary structure of isocitrate lyase deduced from the deoxynucleotide sequence. The primary structure is also compared with that of the enzyme from castor bean derived from cDNA sequencing (1).

Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L.

“…This percent sequence identity is significantly above the 25% level considered to indicate homology between proteins (Doolittle, 1986). Moreover, the 6. napus and Escherichia coli (Matsuoka and McFadden, 1988) polypeptides are 38% similar.…”

“…This percent sequence identity is significantly above the 25% level considered to indicate homology between proteins (Doolittle, 1986). Moreover, the 6. napus and Escherichia coli (Matsuoka and McFadden, 1988) polypeptides are 38% similar.Knowledge of the primary structure of isocitrate lyase may aid in defining structural determinants responsible for targeting the polypeptide to glyoxysomes/peroxisomes. Many peroxisomal proteins, including isocitrate lyase, are not detectably processed during posttranslational import into the organelle, suggesting that the sequences involved in targeting must be located within the mature polypeptide Roberts and Lord, 1981;Lazarow and Fujiki, 1985;Borst, 1986).…”

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

“…This quaternary structure is also described for isocitrate lyases from bacterial and eukaryotic sources. The bacterial isocitrate lyases generally have a 33% lower molecular mass than their eukaryotic counterparts [17]. This is caused by a linker region between domains I and II in the eukaryotic isocitrate lyases.…”

Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris

“…Cleavage sites for restriction enzymes are underlined. The amplification was carried out for 30 cycles in a DNA thermal cycler 2400 (Perkin-Elmer) in a 50 ml reaction mixture containing 100 ng plasmid pCM477 (Watanabe et al, 2002a) or pICL1 (Matsuoka & McFadden, 1988), in turn containing the C. maris icl and E. coli aceA genes, respectively, as template DNA. The reaction mixture also contained 1?5 pmol of each sense and antisense primer, and 1 U of KOD-plus DNA polymerase (Toyobo) in a buffer system provided by the manufacturer.…”