Biotransformation of D-methionine into L-methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Function of several critical amino acids in human pyruvate dehydrogenase revealed by its structure

Pyruvate dehydrogenase (E1), an alpha(2)beta(2) tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of betaW135, alphaP188, alphaM181, alphaH15, and alphaR349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: betaW135 is in the center of the hydrophobic interaction between beta and beta' subunits; alphaP188 and alphaM181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alphaH15 is necessary for the organization of the N-terminus of alpha and alpha' subunits; and alphaR349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.     

The accumulation and metabolism of amino acids by Biomphalaria glabrata, a freshwater pulmonate snail

Freshwater pulmonate snails (Biomphalaria glabrata), pre-treated under bacteriostatic conditions, were incubated in 10 ml of standard medium containing various U-¹⁴C-labelled amino acids at concentrations of 10 μM. Measurements of mass-specific accumulation rates (MSARs) based on HPLC and the accumulation of U-¹⁴C-labelled amino acids into snail tissues have shown unequivocally for the first time that freshwater snails achieved a net accumulation of all the amino acids tested, including aminoisobutyrate (AIB), aspartate, alanine and a mixture of 13 amino acids. There were no significant differences between the MSAR values determined by HPLC from those based on the use of radiolabelled amino acids, whereas MSAR values for control snails were negligible and significantly less. Incubation of snails in media containing radiolabelled aspartate and a mixture of amino acids showed that the accumulated amino acids were readily distributed through the snail’s tissues and then metabolized. The ecological and biochemical questions arising from the fact that freshwater snails are capable of net accumulation of exogenous amino acids at naturally occurring concentrations and subsequent metabolic conversion, contrary to widely held views, are addressed.     

30种植物叶蛋白中氨基酸组成及含量的测定与营养价值评价

评价植物叶蛋白的营养价值,既要考虑“量”,看植物叶蛋白含量的高低,也要考虑“质”,看必需氨基酸的含量及配比[1]。作者取叶蛋白含量50%以上的30种植物进行氨基酸含量测定[2],并进行营养价值评价。1　方　　法应用日立83550型氨基酸自动分析仪进行氨基酸含量的测定,并与?..     

Simultaneous analysis of enantiomeric composition of amino acids and N-acetyl-amino acids by enantioselective chromatography

Among the three chiral columns, CHIROBIOTIC T, CHIRLPAK WH, and CHIRALCEL OD-R, tested for the separation of racemic amino acids and N-acetyl-amino acids, only CHIROBIOTIC T chiral column which is based on covalently bonded amphoteric glycopeptide, teicoplanin, as the stationary phase ligand could be successfully developed to enantiomerically separate racemic amino acids and N-acetyl amino acids simultaneously. This method can be used to determine the enantiomeric composition of amino acids and N-acetyl-amino acids in the catalysis of D-aminoacylase or L-aminoacylase and the conversion rate of N-acylamino acid racemases.     

Micro-analysis of amino acids

An automated amino acid analyzer has been developed for the analysis of amino acids with the sensitivity at the 10–100 pmol level except for proline which requires >50 pmol. o-Phthalaldehyde, in the presence of 2-mercaptoethanol, is used for the fluorometric detection of amino groups (Roth, M. (1971) Anal. Chem. 43, 880–882). A post-column reaction of the amino acid with sodium hypochlorite (Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313–321) gives oxidation products amenable to detection with o-phthalaldehyde. The instrument uses high-performance liquid chromatographic pumps capable of micro-flow rates with a minimum pulsation. The method is suitable for routine analyses of amino acids at picomole levels with reproducibility and accuracy comparable to the ninhydrin-based amino acid analysis.     

Free amino acids in Phycomyces blakesleeanus during development

In Phycomyces blakesleeanus the total level of free amino acids and their relative distributions change strikingly during the development of sporangiophores. Very high levels of serine, glutamate and histidine are found. The variety of amino acids becomes more restricted in sporangiophores grown on nitrogen-rich media; levels of arginine increase dramatically. The distribution differs in mycelia. No unusual amino acids or oligopeptides were found.     

YddG from Escherichia coli promotes export of aromatic amino acids

The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of l-phenylalanine and aromatic amino acid analogues, dl-p-fluorophenylalanine, dl-o-fluorophenylalanine and dl-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of l-phenylalanine, l-tyrosine or l-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. colil-phenylalanine-producing strain containing overexpressed yddG accumulated less l-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.     

Increasing dispensable amino acids in diets of kittens fed essential amino acids at or below their requirement increases the requirement for arginine

Summary Kittens fed diets containing 0.75 × the NRC (1986) essential amino acid requirement (EAArq) and 210 to 560g crude protein(CP)/kg diet exhibited, with increasing CP: 1) decreasing weight gain, 2) decreasing plasma arginine concentrations, 3) increasing urinary orotic acid excretion, 4) increasing plasma glutamic acid concentrations, and 5) plasma isoleucine concentrations at levels that suggest a marginal isoleucine deficiency. Kittens fed a control diet (CD) containing 1.5 × EAArq and 350 g CP/kg diet had maximal weight gains and no orotic aciduria. It was concluded that the decreased weight gain and adverse metabolic effects were caused by arginine deficiency and possibly glutamic acid toxicity induced by high dietary dispensable amino acids. Kittens fed the diets containing 1.0 × EAArq and 350 and 560 g CP/kg diet had depressed plasma arginine and elevated glutamic acid concentrations and orotic aciduria. These results indicate that 10 g arg/kg diet is not adequate at CP concentrations above 280 g/kg and the calculated requirement of arginine is (0.02 g arginine/g CP) × (Y g CP/kg diet) + (4.0 g arginine/kg diet) where Y is the dietary CP level.Abbreviations CD control diet - CP crude protein (g CP/kg diet = g nitrogen/kg diet × 6.25) - DAA dispensable amino acids - EAA essential amino acids - EAArq essential amino acid requirement     

施氮水平对小麦籽粒发育过程中氨基酸含量的影响

施氮能提高小麦籽粒蛋白质氨基酸的含量,并与施氮水平呈正相关;但对普通小麦必需氨基酸与蛋白质氨基酸的比值没有影响,而硬粒小麦4286随施氮水平的提高,该比值下降。在开花后32d以前,籽粒发育过程中游离氨基酸与施氮水平呈正相关,以后,籽粒中游离氨基酸趋于相近,表明施氮增加了游离氨基酸的库源,不同基因型小麦对施氮水平的反应不同,在同等施氮水平和栽培条件下,籽粒中蛋白质氨基酸和游离氨基酸含量为硬粒小麦4286＞小偃6号＞小偃107,不同施氮水平下,籽粒中氨基酸含量为高氮＞中氮＞低氮。     

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.     

Molybdenum Cofactor-Containing Oxidoreductase Family in Plants

Recent investigations on plant molybdenum-containing enzymes that include xanthine dehydrogenase (EC 1.1.1.204) and xanthine oxidase (EC 1.1.3.22), nitrate reductase (EC 1.7.1.1-3), aldehyde oxidase (EC 1.2.3.1), and sulfite oxidase (EC 1.8.3.1) are reviewed. The enzymes belong to closely related protein family and share common structural features. Special attention is being paid to the recently solved crystal structures their implications for the substrate binding and catalytic mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.     

2,4-2硝基氟苯柱前衍生法测定氨基酸的改进

本文报道了用2,4-二硝基氟苯柱前衍生反相高效液相色谱法测定氨基酸,15种常见的氨基酸可得到很好的分离。衍生物很稳定,在室温下保存两个月无明显变化,检测极限可达到5pmol。本法可应用于食品、药品等样品的氨基酸测定。     

An amperometric biosensor for determining amino acids using a bienzymatic system containing amino acid oxidase and protease

An amperometric biosensor for rapid determination of the concentration of l-amino acids has been developed using l-amino acid oxidase (l-AAO) immobilized by gel entrapment with poly(carbamoyl) sulfonate hydrogel. The broad substrate range of l-AAO allows this biosensor to be flexible in application. The artificial sweetener, aspartame, was determined by coupling l-AAO with pronase.     

New branched amino acids for high affinity dendrimeric DC-SIGN ligands

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized.Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido functional groups. As an example of application, d-mannose and l-fucose residues were linked through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with DC-SIGN using SPR competition assay.