Abstract
BackgroundThe growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25°C, forms considerable aggregates at 37°C and completely aggregates at 44°C.ResultsWe extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44°C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37°C) and elevated (44°C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone.ConclusionsA collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains.
Highlights
The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway
Mutant MetAs enable E. coli growth at elevated temperatures Previously, we identified two amino acid substitutions, I229T and N267D, which conferred stability to the MetA protein [11]
The metA mutations that resulted in the corresponding amino acid substitutions Q96K, L110V, I124L, R160L, A195T, A200E, D218G and F247Y were integrated into the E. coli JW3973 (ΔmetA) chromosome to yield the strains K96, V110, L124, L160, T195, E200, G218 and Y247, respectively
Summary
The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. Mutations that resulted in the amino acid substitutions I229T and N267D enabled the E. coli strain WE to grow at higher temperatures and increased the ability of the strain to tolerate acidic conditions. We extended our stabilization studies using a computer-based design and consensus approach [12] to identify additional mutations that might stabilize the inherently unstable MetA enzyme. The consensus concept approach for engineering thermally stable proteins is based on an idea that by multiple sequence alignment of the homologous counterparts from mesophiles and thermophiles, the nonconsensus amino acid might be determined and substituted with the respective consensus amino acid, contributing to the protein stability [12]. I-Mutant2.0 is a support vector machine-based web server for the automatic prediction of protein stability changes with single-site mutations (http://gpcr.biocomp.unibo. it/~emidio/I-Mutant2.0/I-Mutant2.0_Details.html)
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