Norleucine accumulation by a norleucine-resistant mutant of Serratia marcescens.

A norleucine-resistant mutant was derived from an isoleucine-valine auxotroph of a leucine accumulator of Serratia marcescens. The norleucine-resistant mutant could accumulate norleucine from norvaline in the medium without the addition of methionine, which antagonized norleucine. This mutant constitutively formed homoserine-O-transsuccinylase.     

Derepression and altered feedback regulation of valine biosynthetic pathway in analogue-resistant mutants of Streptomyces cinnamonensis resulting in 2-ketoisovalerate excretion

Excretion of 2-ketoisovaleric acid (KIV) was demonstrated in Streptomyces cinnamonensis mutants resistant to valine analogues 2-amino-3-chlorobutyrate, 2-aminobutyrate and norleucine, respectively. The highest KIV concentrations of 170–230 mg l⁻¹ were found in cultivation liquids of norleucine-resistant strains. Biochemical analyses of the acetohydroxyacid synthase (AHAS), valine dehydrogenase (VDH) and branched chain amino acid aminotransferase activities revealed the deregulation of the valine-synthesizing pathway, resulting in KIV excretion. In the 2-amino-3-chlorobutyrate-resistant strain, the activity of AHAS increased 23- to 31-fold compared with the parental strain. The norleucine-resistant mutants combined both a 10- to 23-fold increase in AHAS activity and lack of efficient feedback regulation by valine. In the double 2-amino-3-chlorobutyrate plus norleucine-resistant mutant, the AHAS activity was only four to eight-fold higher, but release of feedbackregulation was conserved. Similarly, feedback regulation was inefficient in 2-aminobutyrate-resistant strains, however the AHAS activity was lower than in the parental strain. A strong induction of VDH was observed in all regulatory mutants.     

Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

Overproduction of noncanonical amino acids by <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E. coli

Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E. coli have been detected. Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC. The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry. In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E. coli.     

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.     

Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase

Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine. Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme. Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.     

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.     

The fate of norleucine as a replacement for methionine in protein synthesis.

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNA_met species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-³H]norleucine and [³⁵S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNA^f_met. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNA^f_met can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNA^m_met, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine.     

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:204–211, 2015     

Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

Regulatory mutants of Streptomyces cinnamonensis producing monensin A

Abstract We prepared mutants of Streptomyces cinnamonensis resistant to amino acid analogues: 2-aminobutyrate, norvaline, norleucine, 2-amino-3-chlorobutyrate and ethionine. The regulatory mutants were studied as to their production of oligoketide antibiotics, monensins A and B, as dependent on the formation of valine which is a precursor of the butyrate building unit of monensin A. Strains resistant to both 2-amino-3-chlorobutyrate and norleucine exhibited an increased production of monensin A from 50% to 90–93% of total monensins.     

Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats

Acute administration of leucine and norleucine activates the mammalian target of rapamycin (mTOR) cell-signaling pathway and increases rates of protein synthesis in a number of tissues in fasted rats. Although persistent stimulation of mTOR signaling is thought to increase protein synthetic capacity, little information is available concerning the effects of chronic administration of these agonists on protein synthesis, mTOR signal transduction, or leucine metabolism. Hence, we developed a model of chronic leucine/norleucine supplementation via drinking water and examined the effects of chronic (12 days) supplementation on protein synthesis in adipose tissue, kidney, heart, liver, and skeletal muscle from ad libitum-fed rats. The relative concentration of proteins involved in mTOR signaling and the two initial steps in leucine oxidation were also examined. Leucine or norleucine supplementation was accompanied by increased rates of protein synthesis in adipose tissue, liver, and skeletal muscle, but not in heart or kidney. Supplementation was not associated with increases in the anabolic hormones insulin or insulin-like growth factor I. Chronic supplementation did not cause apparent adaptation in either components of the mTOR cell-signaling pathway that respond to leucine (mTOR, ribosomal protein S6 kinase, and eukaryotic initiation factor 4E-binding protein-1) or the first two steps in leucine metabolism (the mitochondrial isoform of branched-chain amino acid transaminase, branched-chain keto acid dehydrogenase, and branched-chain keto acid dehydrogenase kinase), which may be involved in terminating the signal from leucine. These results suggest that provision of leucine or norleucine supplementation via the drinking water results in stimulation of postprandial protein synthesis in adipose tissue, skeletal muscle, and liver without notable adaptive changes in signaling proteins or metabolic enzymes.     

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1). A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.     

Definitive evidence for similarity in the active site of renin and acidic protease

The pressor enzyme renin from the mouse submaxillary gland was inactivated rapidly by diazoacetyl-D,L-norleucine methyl ester in the presence of cupric ion. The ion was essential to this reaction. The complete inactivation was obtained by the stoichiometric reaction of the reagent as indicated by the stoichiometric incorporation of norleucine into the renin molecule. The incorporated norleucine could be removed by treatment with 0.2 M hydroxylamine in 8 M urea indicating that a carboxyl group presumably essential for catalysis was esterified by the aliphatic diazo reagent. Renin from the hog kidney was also inactivated completely by a similar reaction. Since the requirement of cupric ion in the inactivation by aliphatic diazo compound is the most notable specific feature of acidic proteases, it is proposed that renin have a catalytic site similar to those of acidic proteases and that renin belongs to the family of acidic proteases, though its specificity determining site may be somewhat different.     

Isolation and characterization of norleucine-resistant baby hamster kidney cells

Variants resistant to low and high levels of the methionine analogue norleucine were isolated in baby hamster kidney cells and in two clonal sublines, B1 and TG2. Clones resistant to high levels of norleucine were observed only after chemical mutagenesis, whereas clones capable of growing in low concentrations of norleucine occurred with equal frequency spontaneously and after mutagenesis. The variants were characterized with respect to uptake of ¹⁴C-norleucine and ¹⁴C-methionine. Five clones were found to be deficient in ¹⁴C-norleucine uptake, and of these, four showed reduced ¹⁴C-methionine uptake. The variants were tested also for increased activity of N₅-methyl-tetrahydrofolate: homocysteine methyltransferase, the enzyme which catalyses the terminal reaction in methionine biosynthesis. In four clones, higher levels of the methyltransferase were present than in the wild-type cells, suggesting overproduction or stabilization of this enzyme.