A functional leuABCD operon is required for leucine synthesis by the tyrosine-repressible transaminase in Escherichia coli K-12

Abstract

In Escherichia coli K-12, two enzymes, encoded by ilvE and tyrB, catalyze the amination of 2-ketoisocaproate (2-KIC) to form leucine. Although leucine-requiring derivatives of an ilvE strain that are unable to grow on 2-KIC were expected to have mutations only in tyrB, mapping studies showed that one such mutation was tightly linked to the leu operon (at 1.5 min), not to tyrB (at 92 min). Chromosomal fragments cloned because they complemented this mutation were found to complement leu mutations, and vice versa, but none of these fragments complemented a tyrB mutation. The TnS insertion and flanking host DNA from this anomalous mutant was cloned in vivo, using Mu d14042, and an in vivo procedure was developed to isolate deletion derivatives of Tn5-containing plasmids. These deletion plasmids were used to determine the DNA sequences flanking the transposon. The data showed that TnS was inserted between bp 122 and 132 in the leu leader. In addition, other ilvE leu double mutants were found to be unable to grow on 2-KIC in place of leucine. The accumulation of 2-ketoisovalerate in ilvE lu double mutants was shown to interfere with 2-KIC amination by the tyrB-encoded transaninase and also by the aspC-and avl4-encoded transaminases (which are able to catalyze this reaction in vivo when the corresponding genes are present on multicopy plasmids).In wild-type strains of Escherichia coli, the first three steps in leucine biosynthesis are catalyzed by enzymes encoded by the leuABCD operon, while the last step, the amination of 2-ketoisocaproate (2-KIC) to leucine, is catalyzed by two independent enzymes: the branched chain amino acid transaminase (transaminase B, TrB), encoded by dyE, and the tyrosine-repressible transaminase (transaminase D, TrD), encoded by tyrB (3) (Fig. 1). leuA, leuB, leuC, and leuD mutant strains can grow on either leucine or 2-KIC, while ilvE tyrB double mutants can grow on leucine but not on 2-KIC and ilvE or tyrB single mutants do not require either metabolite (10).The transaminases encoded by two other genes, aspC (TrA) and avtA (TrC), do not catalyze physiologically detectable leucine synthesis when present in a single copy in the chromosome, but when either gene is present on a multicopy plasmid, the leucine requirement of an ilvE tyrB mutant is suppressed. Thus, when overproduced, these two transaminases can also catalyze leucine synthesis in vivo (7).Here we describe the isolation, characterization, and mapping of a TnS-induced presumptive tyrB mutation in an ilvE strain. Although the mutation conferred an absolute requirement for leucine that could not be satisfied by 2-KIC, which is the phenotype expected of an ilvE tyrB double mutant, the Tn5 insertion was found to be in the leu leader region. Other ilvE leu double mutants also were unable to grow on 2-KIC. The data indicate that the inability of ilvE leu double mutants to grow on 2-KIC is due to intracellular 2-ketoisovalerate (2-KIV) accumulation, which has an inhibitory effect on TrD in leucine synthesis. * Corresponding author.

The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine

“…Alternatively, we suppose that a secondary side reaction of the C-S lyase was detected, since the enzymatic activity with substrate amino acids was due to the multiple copies of the corresponding aecD gene. Similar phenomena for catalytic activities of enzymes on additional substrates upon amplification of the corresponding wild-type gene have been reported by several authors (8,11,66).…”

Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties

“…Therefore, we hypothesise that ancestral enzymes of increasing evolutionary age may be imposing a fitness cost on modern bacteria due to an increasing degree of uncoupling between the ancestral LeuB and the other enzymes of the leucine biosynthetic pathway. Biosynthesis of leucine begins with an intermediate from the valine biosynthetic pathway that is processed by LeuA (IPM synthase), LeuB, LeuCD (IPM isomerase) and an aminotransferase into leucine (Vartak et al 1991). In E. coli and B. subtilis, this pathway is regulated via two mechanisms: feedback inhibition of LeuA by leucine, and leucine-mediated transcriptional attenuation of the leuABCD operon (Freundlich et al 1962;Ward and Zahler 1973).…”

H‐NS‐mediated repression of CRISPR‐based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO

“…One of these proteins is the regulator LeuO (Chen and Wu, 2005; De la Cruz et al ., 2007), which belongs to the LysR family of transcription factors (Stoebel et al ., 2008) and is found in all proteobacteria, except the δ subdivision (Maddocks and Oyston, 2008). The leuO gene maps next to the leuABCD operon (Hertzberg et al ., 1980; Chen and Wu, 2005; Chen et al ., 2005), whose gene products are required for leucine synthesis (Vartak et al ., 1991). Recent data indicate that LeuO is involved in regulating transcription of many genes, often as an H‐NS antagonist (Stoebel et al ., 2008; Shimada et al ., 2009).…”