A theoretical elucidation of bilirubin interaction with HSA's lysines: first electrostatic binding site in IIA subdomain

The electrostatic interaction of amino acid lysines 190, 195 and 199 of human serum albumin (HSA) with bilirubin have been investigated using molecular dynamic simulations, QM and QM/MM minimization methods. In this study two methodological approaches have been employed. In the first approach X-ray structure and the structure obtained from the molecular dynamic simulation of subdomain IIA of HSA in vacuum have been utilized. Interactions have been evaluated with the segment 186-200 of the cited subdomain. Calculations on the X-ray structure of above segment indicate an effective interaction of the lysine 195 with bilirubin, although that of the lysine 190 is also found considerable in this structure. Performing simulation in vacuum, it has been revealed that except for the lysine 195, the other two lysine residues (190 and 199) could not be considered as centers of interaction. Such finding, which is in accord with experimental data, lends support to the procedure employed in this study. NBO analyses suggest that tasks to achieve a structure indicating bilirubin interaction with the lysine 195 from the 186-200 segment extracted from X-ray structure, results in a structure that lacks any electrostatic interaction. In fact, it has been found that the stability of the latter species can be attributed to the H-bonding interaction of the glutamate 188 with both bilirubin and the lysine 195. Further NBO analysis on the structure of the same species, while achieved after molecular dynamic simulation on subdomain IIA in vacuum has revealed that a favorable electrostatic interaction between the lysine 195 and bilirubin has occurred. Besides, H-bonding interaction of the glutamate 188 with bilirubin has been evident in the same species. For the second approach, presence of water molecules and ions has been considered to simulate condensed medium. Applying docking, conformational sampling, and QM/MM minimization steps in sequence, a structure has been achieved which presents a specific interaction between epsilon-NH3(+) group of the lysine 195 residue and the lactam oxygen atom of bilirubin. NBO analyses suggest that above electrostatic interaction is combined with hydrogen bonding interaction between same two groups. Moreover, a hydrogen bond between oxygen atom of bilirubin's acetate group and alpha-NH group of lysine 195 has been observed. Molecular orbital calculations have been presented which support the NBO analyses.     

L—赖氨酸高产菌株选育的研究

L-赖氨酸产生菌钝齿棒杆菌（Corynebacteriumcrenatum）N30－25菌株经紫外线诱变处理,分别在含有不同浓度的七叶苷的培养基上进行筛选,经摇瓶多次复筛获得了3株高产变异菌株。对这3株菌在相同发酵条件下进行发酵生产L－赖氨酸,与出发菌株比较,产量提高了22－31％,经过3次传代,产生L－赖氨酸能力仍很稳定。     

Lysine biosynthesis in microbes: relevance as drug target and prospects for β-lactam antibiotics production

Plants as well as pro- and eukaryotic microorganisms are able to synthesise lysine via de novo synthesis. While plants and bacteria, with some exceptions, rely on variations of the meso-diaminopimelate pathway for lysine biosynthesis, fungi exclusively use the α-aminoadipate pathway. Although bacteria and fungi are, in principle, both suitable as lysine producers, current industrial fermentations rely on the use of bacteria. In contrast, fungi are important producers of β-lactam antibiotics such as penicillins or cephalosporins. The synthesis of these antibiotics strictly depends on α-aminoadipate deriving from lysine biosynthesis. Interestingly, despite the resulting industrial importance of the fungal α-aminoadipate pathway, biochemical reactions leading to α-aminoadipate formation have only been studied on a limited number of fungal species. In this respect, just recently an essential isomerisation reaction required for the formation of α-aminoadipate has been elucidated in detail. This review summarises biochemical pathways leading to lysine production, discusses the suitability of interrupting lysine biosynthesis as target for new antibacterial and antifungal compounds and emphasises on biochemical reactions involved in the formation of α-aminoadipate in fungi as an essential intermediate for both, lysine and β-lactam antibiotics production.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S-adenosylmethionine and S-2-aminoethyl- l-cysteine on the lysine 2-oxoglutarate reductase-saccharopine dehydrogenase bifunctional enzyme

Lysine biosynthesis has been extensively studied and the regulatory enzymes characterized in some of the most important crop plants, however, much less is known about the lysine degradation pathway. Lysine 2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase (SDH) have recently been partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have further characterized these enzymes from rice endosperm in relation to Ca²⁺ and ionic strength modulation. Optimum pH values of 7.0 and 8.0 were obtained for LOR and SDH, respectively. The LOR domain of the polypeptide was modulated by Ca²⁺ and ionic strength, whereas the SDH domain was not. It would appear that the modulation by Ca²⁺ and ionic strength of LOR is a common feature among plant LOR enzymes. S -adenosylmethionine (SAM) did not produce any significant effect on either enzyme activity, indicating that it only plays a role in the regulation of lysine biosynthesis. The effect of S -2-aminoethyl- l -cysteine (AEC) as both a substrate and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for LOR, but was also able to inhibit LOR activity, possibly competing with lysine at the active site. The higher K_m for AEC compared to lysine may reflect a lower binding affinity for AEC.     

Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

Intracellular transport of thialysine and selenalysine in CHO cells

The intracellular transport of thialysine and selenalysine in CHO cells has been studied. Data have been obtained indicating that the two lysine analogs can be transported by both the cationic aminoacid transport system and by the L transport system. The affinity of the cationic aminoacid transport system is similar for the two lysine analogs but lower than that for lysine and the affinity of the L transport system for the two lysine analogs is lower than that for leucine.     

A bioluminescent Escherichia coli auxotroph for use in an in vitro lysine availability assay

Microbiological methods have been used to determine the amino acid availability of a variety of animal feed and human food protein sources. Growth of Escherichia coli auxotrophs have been shown to yield a consistent linear response to lysine concentration when compared to chemical measures. Extent of total growth of E. coli lysine mutant (American Type Culture Collection #23812) when measured as optical density (OD) displays a lysine-dependent growth response that can be used to estimate lysine in feed proteins. However, typical OD-based growth studies for amino acid quantitation using the mutant may require anywhere from 12 to over 40 h. To develop an improved rapid method for lysine quantitation in protein sources, the plasmid pJHD500 carrying genes that encode for expression of bioluminescence and ampicillin resistance was transformed into the E. coli mutant by electroporation (set at 1.80 kV). The luminescence measured during early exponential growth allowed detectable differentiation of lysine concentration in the media in 4 h. When the luminescence method was compared with the conventional optical density lysine growth assay, the correlation coefficient was 0.989. Lysine availability valued for enzymatically hydrolyzed protein sources were comparable with availability measures using animal methods for lysine availability. This research shows potential applications for more rapid quantitative measurement of bioavailable lysine.     

稻米赖氨酸快速测定条件的探讨

以氨基酸自动分析仪测定稻米赖氨酸含量为参照标准。在“茚三酮测定赖氨酸含量”(A法)的基础上,对该法中大米蛋白质的提取温度和时间、色温度和时间及茚三酮用量等条件进行了探讨,提出了稻米赖氨酸含量快速测(B法)。提取条件为90℃5分钟,显色条件为90℃20分钟;茚三酮试剂用1 ml。用B法对10个大米样品进行测定,赖氨酸含量与氨基酸分析仪测得的赖含量基本上一致,无显著差异(P>0.05)。     

Genetic Analysis of Lysine Auxotrophs of Staphylococcus aureus

The genetics of lysine biosynthesis in Staphylococcus aureus was examined by a transductional analysis of lysine auxotrophs. These mutants had previously been grouped according to their biochemical characteristics. The mutant sites appeared to be closely linked. Complementation was observed between different groups but not between mutant strains belonging to the same group. A strain was detected which seemed to have a mutant control region. Evidence is presented to support the hypothesis that the lysine biosynthetic region functions as an operon.     

Chemical biology and pharmacology of histone lysine methylation inhibitors

Histone lysine methylation is a post-translational modification that plays a key role in the epigenetic regulation of a broad spectrum of biological processes. Moreover, the dysregulation of histone lysine methyltransferases (KMTs) has been implicated in the pathogenesis of several diseases particularly cancer. Due to their pathobiological importance, KMTs have garnered immense attention over the last decade as attractive therapeutic targets. These endeavors have culminated in tens of chemical probes that have been used to interrogate many aspects of histone lysine methylation. Besides, over a dozen inhibitors have been advanced to clinical trials, including the EZH2 inhibitor tazemetostat approved for the treatment of follicular lymphoma and advanced epithelioid sarcoma. In this Review, we highlight the chemical biology and pharmacology of KMT inhibitors and targeted protein degraders focusing on the clinical development of EZH1/2, DOT1L, Menin-MLL, and WDR5-MLL inhibitors. We also briefly discuss the pharmacologic targeting of other KMTs.     

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

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.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics

Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.     

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives

l-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like l-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.