Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12

Berg, Claire M.; Wang, Ming-der; Vartak, Narendra B.; Liu, Lin

doi:10.1016/0378-1119(88)90456-8

Cited by 30 publications

(19 citation statements)

References 25 publications

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“…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).…”

Section: Discussionsupporting

confidence: 87%

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

Rossol

Pühler

1992

J Bacteriol

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S-(beta-Aminoethyl)-cysteine (AEC) resistance was achieved in Corynebacterium glutamicum by cloning a chromosomal 1.5-kb EcoRV-BglII DNA fragment on a multicopy plasmid. DNA sequence analysis of the 1.5-kb DNA fragment revealed an open reading frame (ORF326) which represents the AEC resistance gene, designated aecD. The aecD gene directs the synthesis of a 36-kDa protein which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aecD gene is a nonessential gene and mediates AEC resistance only in an amplified state. C. glutamicum strains harboring an amplified aecD gene can utilize AEC as an alternative nitrogen source, indicating that the AEC resistance mechanism is due to AEC degradation. Since the AEC degradation products analyzed by high-pressure liquid chromatography were found to be pyruvate and aminoethanethiol (cysteamine), it was concluded that the aecD gene encodes a C-S lyase with alpha, beta-elimination activity. Besides AEC, the C-S lyase was also able to use cysteine, cystine, and cystathionine as substrates.

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Section: Discussionsupporting

confidence: 87%

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

Rossol

Pühler

1992

J Bacteriol

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“…There is ample precedent that both IlvA (26,35,71) and IlvE (22,49) interact with molecules other than their substrates in the Ile biosynthetic pathway. In fact, the promiscuity of transaminases (and deaminases) in general has been well documented (2,3,37,38,43,49,64,65,68). Further, multiple enzymes have been shown to catalyze side reactions that can be critical for metabolic processes and/or growth phenotypes in the appropriate strain background (7,45,54,57).…”

Section: Discussionmentioning

confidence: 99%

Reduced Transaminase B (IlvE) Activity Caused by the Lack ofyjgFIs Dependent on the Status of Threonine Deaminase (IlvA) inSalmonella entericaSerovar Typhimurium

2004

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The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.

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“…Cells were grown overnight at 300C in appropriately supplemented glucose medium E, and crude extracts were prepared and assayed as described previously (7), except that the appropriate antibiotic was added to the medium for plasmid-containing strains. Leucine transamination was assayed in a coupled reaction using L-glutamate as the amino donor and 2-KIC as the amino acceptor (7,10 one experiment, crude extracts were purified on a Sephadex G-25 column.…”

mentioning

confidence: 99%

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

et al. 1991

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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.

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Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12

Cited by 30 publications

References 25 publications

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

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

Reduced Transaminase B (IlvE) Activity Caused by the Lack ofyjgFIs Dependent on the Status of Threonine Deaminase (IlvA) inSalmonella entericaSerovar Typhimurium

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

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