Regulation of homoserine O-succinyltransferase for efficient production of L-methionine in engineered Escherichia coli.

l-Methionine biosynthesis in Eschericha coli consists of multiple unit modules with various enzymes involved and the imbalance between different modules always restricted its productivity. In this study, the key enzymes participating in the pathway were investigated for their effect on l-methionine production and the pivotal enzyme homoserine O-succinyltransferase (MetA) was designed to be regulated. The surface amino acid residues of MetA were effectively modified through site-saturation mutagenesis and single mutants L63F, A28V, P298L and double mutant L63F/A28V were obtained with improved l-methionine productivity. The structure analysis revealed that the involved residues were on the surface loop regions, which was proposed to be conducive to the refolding of MetA and thus reduce the inhibition effect caused by l-methionine. After expression of the selected single mutant L63F in engineered E. coli ΔIJA-HFEBC strain with l-methionine efflux pump and mutated 3-phosphoglycerate dehydrogenase, the l-methionine production was significantly improved, with a final yield of 3528 mg/L. The results demonstrated the efficiency of MetA regulation for enhanced production of l-methionine and meanwhile provided important guidance for further engineering of MetA with increased l-methionine productivity.

Stabilization of homoserine-O-succinyltransferase (MetA) decreases the frequency of persisters in Escherichia coli under stressful conditions.

Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in Escherichia coli cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native metA gene on the E. coli K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in E. coli K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.

Stabilized homoserine o-succinyltransferases (MetA) or L-methionine partially recovers the growth defect in Escherichia coli lacking ATP-dependent proteases or the DnaK chaperone.

BACKGROUND: 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. 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. RESULTS: We 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. CONCLUSIONS: A 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.

Methionine biosynthesis in Agrobacterium tumefaciens: study of the first enzyme.

Here we characterize the first step in methionine biosynthesis in Agrobacterium tumefaciens, an α-proteobacterium. We explored the metA gene and its products and found several unique properties. Although the gene was annotated as a homoserine transsuccinylase, based upon sequence similarity to characterized homologs in other bacteria, including Escherichia coli, the enzyme uses acetyl-CoA as a substrate and therefore is functionally a transacetylase. Moreover, the protein is thermolabile and the gene is under regulation of heat shock transcriptional activator σ32. 3. The gene has a SAM-riboswitch, which shuts off transcription by σ-32 as well as by the vegetative σ-70.

E. coli transports aggregated proteins to the poles by a specific and energy-dependent process.

Aggregation of proteins due to failure of quality control mechanisms is deleterious to both eukaryotes and prokaryotes. We found that in Escherichia coli, protein aggregates are delivered to the pole and form a large polar aggregate (LPA). The formation of LPAs involves two steps: the formation of multiple small aggregates and the delivery of these aggregates to the pole to form an LPA. Formation of randomly distributed aggregates, their delivery to the poles, and LPA formation are all energy-dependent processes. The latter steps require the proton motive force, activities of the DnaK and DnaJ chaperones, and MreB. About 90 min after their formation, the LPAs are dissolved in a process that is dependent upon ClpB, DnaK, and energy. Our results confirm and substantiate the notion that the formation of LPAs allows asymmetric inheritance of the aggregated proteins to a small number of daughter cells, enabling their rapid elimination from most of the bacterial population. Moreover, the results show that the processing of aggregated proteins by the protein quality control system is a multi-step process with distinct spatial and temporal controls.

Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase.

In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44 degrees C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA(E. coli) protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44 degrees C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA(E. coli) to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA(E. coli) enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis.

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.

Substrate analysis of homoserine acyltransferase from Bacillus cereus.

Substrate specificity within the family of enzymes designated as homoserine transsuccinylases is variable, with some organisms utilizing succinyl-CoA and other organisms utilizing acetyl-CoA. In this study it is shown that the enzyme from Bacillus cereus uses acetyl-CoA as its acyl donor, but its catalytic rate is significantly lower than other HTS family members. BcHTS is inactivated by both iodoacetamide and diethyl pyrocarbonate and the enzyme can be partially protected from inactivation by the presence of succinyl-CoA. This leads to the conclusion that BcHTS can bind both acetyl-CoA and succinyl-CoA and suggests that it may represent an intermediate between the succinate-transferring HTS family members and the acetate-transferring HTS family members. The B. cereus enzyme was unable to rescue growth of an Escherichia coli strain lacking a functional transsuccinylase, however.

Tools for the study of protein quality control systems: use of truncated homoserine trans-succinylase as a model substrate for ATP-dependent proteolysis in Escherichia coli.

Protein quality control, mediated by chaperones and ATP-dependent proteases, is essential for maintaining balanced growth and for regulating critical processes. To study these systems it is necessary to have model substrate proteins. However, most cellular proteins are stable and the few unstable proteins are usually regulatory and present in low concentrations, making them unsuitable for studies, especially in vivo. We present HTS(Delta1-6), a truncated homoserine trans-succinylase (HTS) which is unstable, can be expressed at high levels and has an enzymatic, measurable, activity. This protein can serve as a good model substrate for Escherichia coli ATP-dependent proteolysis.

Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.

Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.

Purification and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase.

The methionine biosynthetic pathway found in bacteria is controlled at the first step, acylation of the gamma-hydroxyl of homoserine. This reaction is catalyzed by one of two unique enzymes, homoserine transacetylase or homoserine transsuccinylase, which have no amino acid sequence similarity. We cloned, expressed, and purified homoserine transsuccinylase from the thermophilic bacterium Thermotoga maritima. Substrate specificity experiments demonstrated that acetyl-coenzyme A (CoA) is the preferred acyl donor and is used at least 30-fold more efficiently than succinyl-CoA. Steady-state kinetic experiments confirm that the enzyme utilizes a ping-pong kinetic mechanism in which the acetate group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine. The maximal velocity, V/K (acetyl-CoA) and V/K (homoserine), all exhibited bell-shaped pH curves with apparent pKs of 6.0-6.9 and 8.2-8.8. The enzyme was inactivated by iodoacetamide in a pH-dependent manner, with an apparent pK of 6.3, suggesting the presence of an active-site cysteine residue which forms an acetyl-enzyme thioester intermediate during catalytic turnover, similar to observations with other transsuccinylases. In addition, the enzyme is highly stable at elevated temperatures, maintaining full activity at 70 degrees C. Taken together, these data suggest that the T. maritima enzyme functions biochemically as a transacetylase, despite having the sequence of a transsuccinylase.