Involvement of lysine-193 of the conserved "K-T-G-G" motif in the catalysis of maize starch synthase IIa

It has been suggested that the lysine residue in the conserved K-T-G-G motif could be the substrate ADP-glucose binding site of Escherichia coli glycogen synthase (GS). Since the K-X-G-G motif is highly conserved between E. coli GS and all the maize starch synthase (SS) isozymes, it has become widely accepted that the lysine in the conserved K-T-G-G motif may also function as the ADPGlc binding site of maize SS. We have used chemical modification and site-directed mutagenesis to study the function of lysine residues in SS. Pyridoxal-5'-phosphate inactivated maize SSIIa activity in a time and concentration dependent manner. ADPGlc completely protected SSIIa from inactivation by pyridoxal-5'-phosphate, indicating that lysine residue(s) could be important for ADPGlc binding and enzyme catalysis. In contrast to E. coli GS, mutation of conserved lysine193 (K-T-G-G) in maize SS did not alter the ADPGlc binding while significantly changing the enzyme activity toward different primers. Our results suggest that lysine-193 (K-T-G-G) is not directly involved in ADPGlc binding, instead mutation in the conserved lysine position affected the primer preference.     

Evidence for the genetic control of lysine catabolism in maize endosperm

A split pollination was used to produce normal (Su su su O2 o2 o2) and high lysine double mutant sugary opaque-2 (su su su o2 o2 o2) endosperms on the same ear of sugary opaque-2 maize plants. Amino acids were determined in the vascular sap of the ear peduncle. Lysine content in the sap was compared with lysine stored in both normal and sugary opaque-2 endosperm during kernel filling. Lysine content in the ear peduncle sap could account for all lysine found in both endosperms. Preformed lysine is highly catabolized in the normal endosperm, but not in the high lysine sugary opaque-2 endosperm. The rate of lysine breakdown appears to be an important mechanism by which the high lysine mutant controls lysine level in maize endosperm.     

Lysine synthesized by the gastrointestinal microflora of pigs is absorbed,mostly in the small intestine

This study used a digesta transfer protocol to determine the site of absorption of lysine synthesized by the gastrointestinal microflora of pigs. Eight pigs were used, four with reentrant cannulas in the terminal ileum, two with simple T cannulas in the terminal ileum, and two intact. All pigs were given, for 5 days, the same low-protein diet that included fermentable carbohydrates. The diet of two pigs with reentrant cannulas (donor) and of the two intact (control) pigs was supplemented with (15)NH(4)Cl. The two other pigs with reentrant cannulas (acceptor pigs) and those with simple cannulas (used to supply unlabeled digesta) were given the same diet but unlabeled NH(4)Cl. Ileal digesta were collected continuously from all of the reentrant cannulas and kept on ice. All digesta from each donor pig were reheated and returned to the distal cannula of its companion acceptor, whose ileal digesta were discarded. Unlabeled ileal digesta from the pigs with simple cannulas were instilled into the distal cannulas of the donor pigs. At the end of the experiment, the average (15)N enrichment in the plasma free lysine of control pigs was 0.0407 atom % excess (APE); that of donor pigs was 0.0322 APE (79% of controls), whereas that of acceptor pigs was only 0.0096 APE (24% of controls). Due to nitrogen recycling, acceptor pigs had labeled lysine in the digesta of the stomach and small intestine, and donor pigs had labeled lysine in the digesta of the large intestine. If account is taken of the higher (15)N enrichment of microbial lysine in the large compared with the small intestine, it can be estimated that >90% of the absorption of microbial lysine took place in the small intestine.     

Thialysine utilization for protein synthesis by CHO cells

Chinese Hamster Ovary (CHO) cells utilize thialysine when added to the culture medium. Thialysine utilization is prevented by increasing lysine concentration in the medium, thus indicating that thialysine is utilized in substitution for and in competition with lysine. Almost all thialysine disappeared from the medium is recovered in cell protein hydrolysates. Thialysine is used for protein synthesis in substitution for lysine, and up to 10% of lysine can be substituted.     

Design, expression and characterisation of lysine-rich forms of the barley seed protein CI-2

Chymotrypsin inhibitor CI-2 is a small (84 residue) barley seed protein that has been used extensively to study protein folding. It also contains eight lysine residues, making it an attractive target for expression in transgenic plants to increase their lysine contents. We have designed three lysine-enriched forms of CI-2 and compared their structures and properties with that of the wild type protein. One mutant containing three additional lysine residues in the inhibitory loop shows high stability to denaturation and reduced inhibitory activity, indicating its suitability for use in genetic engineering.     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Identification of the last unknown genes in the fermentation pathway of lysine

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.     

A [4,5-3H]lysine:[14C]lysine dual-label method to measure lysine hydroxylation in collagen

A new method has been developed to determine the extent of lysine hydroxylation in newly synthesized collagen. This method relies on the measurement of changes in the ratio of [3H]lysine:[14C]lysine in collagenase digests, resulting from loss of tritium from the C-5 position of lysine during hydroxylation. Lysine hydroxylation can be measured in the presence of large amounts of noncollagen proteins, and simultaneous quantitation of the relative rates of collagen and non-collagen protein production is obtained. The dual-label lysine method is simple, rapid, and accurate. There was a very good correlation between this method and column chromatography procedures currently used for the measurement of lysine hydroxylation.     

C‐terminal lysine processing of human immunoglobulin G2 heavy chain in vivo

Although human IgG heavy chain genes encode a C‐terminal lysine, this residue is mostly absent from the endogenous antibodies isolated from serum. Some low but variable level of C‐terminal lysine is present on therapeutic antibodies expressed in mammalian cell culture systems. Here, we monitored the C‐terminal lysine processing of a recombinant human IgG2 antibody after intravenous injection into human subjects. Peptide mapping of the therapeutic antibody isolated from serum samples by affinity purification was used to quantify the C‐terminal lysine levels over time in vivo. The C‐terminal lysine residue was found to be rapidly lost in vivo with a half life of about an hour (62 min). In vivo C‐terminal lysine processing could be reproduced in vitro, but at a faster rate, by incubating in human serum. Pretreated serum, under conditions used to inactivate carboxypeptidase U, generated in vitro C‐terminal lysine processing rates that more closely matched those in vivo. Endogenous IgG, isolated from human blood, contained very low levels of C‐terminal lysine (～0.02%), consistent with the expected circulating half life of antibodies and the calculated C‐terminal lysine processing rate. Thus, the low residual IgG2 C‐terminal lysine is rapidly processed in vivo and such processing likely occurs on endogenous antibodies in circulation. Biotechnol. Bioeng. 2011;108: 404–412. © 2010 Wiley Periodicals, Inc.     

Structure of a substrate radical intermediate in the reaction of lysine 2,3-aminomutase.

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Global Proteome and Lysine Succinylome Analyses Reveal the Effects of Energy Metabolism in Renal Cell Carcinoma

In light of the increasing incidence of renal cell carcinoma (RCC), its molecular mechanisms have been comprehensively explored in numerous recent studies. However, few studies focus on the influence of multi‐factor interactions during the occurrence and development of RCC. This study aims to investigate the quantitative global proteome and the changes in lysine succinylation in related proteins, seeking to facilitate a better understanding of the molecular mechanisms underlying RCC. LC‐MS/MS combined with bioinformatics analysis are used to quantitatively detect the perspectives at the global protein level. IP and WB analysis were conducted to further verify the alternations of related proteins and lysine succinylation. A total of 3,217 proteins and 1,238 lysine succinylation sites are quantified in RCC tissues, and 668 differentially expressed proteins and 161 differentially expressed lysine succinylation sites are identified. Besides, expressions of PGK1 and PKM2 at protein and lysine, succinylation levels are significantly altered in RCC tissues. Bioinformatics analysis indicates that the glycolysis pathway is a potential mechanism of RCC progression and lysine succinylation may plays a potential role in energy metabolism. These results can provide a new direction for exploring the molecular mechanism of RCC tumorigenesis.     

Lysine flux in dry and lactating dairy goats

The objective of this experiment was to test the hypothesis that the physiological state of lactation is accompanied by both an increase in total plasma lysine flux (rate of loss and replacement of lysine) and a net reduction in flux through the plasma lysine pool after accounting for lysine secreted in the milk. Eight lactating French Alpine does were primed and infused for three hours with solutions of alpha15N L-lysine HCl in 0.9% saline through indwelling jugular vein catheters. Enrichment of circulating plasma lysine by continuous intravenous infusion of alpha15N L-lysine was used to estimate whole body lysine flux. This procedure was repeated one month after cessation of milking. Total plasma lysine flux was similar for dry and lactating does (116.6 and 123.0 mmol/d, SEM 16.6 mmol), but 54.2 mmol/d lysine was secreted as milk protein during lactation. Direct measurement of lysine absorption from the lower tract and independent measurement of lysine degradation are needed to provide a more complete portrait of caprine lysine kinetics.     

Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica

The locations have been determined, with respect to the plasma membrane, of lysine alpha 380 and lysine gamma 486 in the alpha subunit and the gamma subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the alpha subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the gamma subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine alpha 380 and lysine gamma 486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine alpha 380 in the native acetylcholine receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine gamma 486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine alpha 380 is on the inside surface of a vesicle and lysine gamma 486 is on the outside surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of specific lysine modifications to identify the site of reaction between cytochrome c and ferricyanide

The site of the reaction between horse heart ferrocytochrome c and ferricyanide was investigated by measuring the reaction rate of cytochrome c derivatives specifically modified at single lysine residues to form trifluoroacetyl or trifluoromethylphenylcarbamyl amino groups. Cytochrome c derivatives singly modified at lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice had rate constants decreased from that of native cytochrome c by factors of 1.29, 2.03, 1.12, 1.35, 1.46, 1.29, and 1.19, respectively. Modification of a given lysine with the bulky trifluoromethylphenylcarbamyl group caused nearly the same decrease in reaction rate as modification with the trifluoroacetyl group, indicating that the effect was due to removal of an electrostatic interaction between the protonated lysine amino group and ferricyanide. Modification of lysines 22, 55, 99, and 100 at the right side, bottom, and back of cytochrome c had no effect on the reaction rate. These results indicate that the reaction site is located at the exposed edge of the heme and that the electrostatic interaction between ferricyanide and cytochrome c is dominated by the lysine amino groups surrounding the heme crevice, which include lysine 86, in addition to the ones listed above. We have used the specific lysine modification results to estimate the contribution of each lysine amino group to the electrostatic interaction and have developed a semiempirical relation for the total electrostatic interaction.     

Molecular characterization of propionyllysines in non-histone proteins

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones. The molecular components involved in the two protein modification pathways are unknown, hindering further functional studies. Here we report identification of the first three in vivo non-histone protein substrates of lysine propionylation in eukaryotic cells: p53, p300, and CREB-binding protein. We used mass spectrometry to map lysine propionylation sites within these three proteins. We also identified the first two in vivo eukaryotic lysine propionyltransferases, p300 and CREB-binding protein, and the first eukaryotic depropionylase, Sirt1. p300 was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation, like lysine acetylation, is a dynamic and regulatory post-translational modification. Based on these observations, it appears that some enzymes are common to the lysine propionylation and lysine acetylation regulatory pathways. Our studies therefore identified first several important players in lysine propionylation pathway.     

Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Saccharomyces

Hydroxylysine acts as a growth inhibitor of Saccharomyces for a certain period of time. The inhibition is concentration-dependent and is reversed by a small amount of lysine in the medium. After the growth-inhibitory period, the wild-type cells are able to grow rapidly even in the presence of hydroxylysine. Both lysine auxotrophs and wild-type cells are unable to utilize hydroxylysine in place of lysine. Hydroxylysine, mimicking lysine, controls the biosynthesis of lysine and thereby limits the availability of biosynthetic lysine to the cells. Hydroxylysine affects the biosynthesis of lysine at a number of enzymatic steps. Accumulation of homocitric acid, the first intermediate of lysine biosynthesis, in the mutant strains 19B and A B9 is reduced significantly in the presence of hydroxylysine. Hydroxylysine, like lysine, exerts a significant inhibition in vitro on the homocitric acid-synthesizing activity. Enzymes following the alpha-aminoadipic acid step respond in a noncoordinate fashion to hydroxylysine. Level of the enzyme saccharopine reductase, but not of alpha-aminoadipic acid reductase or saccharopine dehydrogenase, is reduced significantly. These regulatory effects of hydroxylysine are similar to those observed for lysine.     

The lysP gene encodes the lysine-specific permease.

Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease.