Hydroxy Amino Acids Research Articles

Oligosaccharyl Transferase is a Constitutive Component of an Oligomeric Protein Complex from Pig Liver Endoplasmic Reticulum

Oligosaccharyl transferase (OST), an intrinsic component of the endoplasmic reticulum membrane, catalyses the N-glycosylation of specific asparagine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/detergent extraction, concanavalin-A precipitation, S-Sepharose, MonoP and concanavalin-A–Sepharose chromatographies. A highly purified OST preparation exerting catalytic activity, contained two protein subunits of 48 kDa and 66 kDa, from which the 66-kDa species was identified by immunoblotting as ribophorin I. The function of ribophorin I in this dimeric protein complex is unknown. The high degree of similarity between its transmembrane region and a putative dolichol-recognition consensus sequence suggests that ribophorin I could be involved in glycolipid binding and delivery. Several lines of evidence indicate that the catalyti-cally active 48-kDa/66-kDa polypeptides are associated in the endoplasmic reticulum membrane with other proteins, including ribophorin II and a 40-kDa glycoprotein. The implication of ribophorins I and II in the translocation machinery and their apparent association with the OST activity point to a close relationship between polypeptide synthesis, translocation and N-glycosylation, both spacially and temporally. Kinetic studies with the MonoP-purified Oligosaccharyl transferase showed that the enzyme transfers dolichyl-diphosphate-linked GlcNAc2 to synthetic tripeptides and hexapeptides, containing the Asn-Xaa-Thr motif, at a comparable rate. The glycosylation reaction was found to have a pH optimum close to 7 and to require divalent metal ions, with Mn2+ being most effective. Substitution of threonine in the N- glycosylation motif by serine impairs its function as an acceptor, measured by Vmax/Km, by approximately 17-fold, consisting of a 7.3-fold increase in Km and a 2.3-fold decrease in Vmax. This indicates that the side chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its participation in the catalytic mechanism of transglycosylation. The Km values of peptide acceptors improved significantly when dolichyl-phosphate-bound oligosaccharides were used instead of lipid-linked GlcNAc2 as the glycosyl donor. We conclude from this observation that the sugar residues on the outer branches of the glycolipid donor induce conformational changes in the active site of the Oligosaccharyl transferase, thus influencing the association constant of the peptide substrate.

ChemInform Abstract: Use of the Aryl Groups as the Carboxyl Synthon. Application to the Synthesis of Some Natural Products Containing Hydroxy Amino Acid Functions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Oligonucleotides and Nucleotide-Peptides. LV. Synthesis and Some Properties of Triesters of Hydroxy Aming Acids and Thymidilyl-(3′ → 5′)-Thymidine1

Abstract N-Cbz-Ser(OMe)-(P1→O)-(TpT) and N-Bz-Tyr(OEt)-(P1→O)-(TpT) were synthesized as diastereomeric mixtures of approximately (1:1) by reacting (TpT) with 2,4,6-triisopropylbezenesulphonyl chloride and hydroxy amino acids Their stability to the action of chemical agents and phosphodiesterases was investgated.

Asymmetric Alkylations of a Sultam‐Derived Glycine Equivalent: Practical preparation of enantiomerically pure α‐amino acids

AbstractAlkylation of the chiral glycine derivative 2 with “activated” organohalides under ultrasound‐assisted phasetransfer catalysis or with activated and nonactivated organohalides in anhydrous medium provides (mostly crystalline) alkylation products 3. Acidic hydrolysis of the pure products 3 gives (aminoacyl)sultams 4 which by mild saponification furnish pure α‐amino acids 5 in good overall yields from 2, along with recovered auxiliary 1 (Scheme 1). Pure ω‐protected α,ω‐diamino acids and α‐amino‐ω‐(hydroxyamino)acids 12–16 are readily accessible from (ω‐haloacyl)sultams 3 via reaction with N‐nucleophiles followed by acidic and basic hydrolyses (Scheme 2). A reliable determination of the enantiomeric purity of α‐amino acids using HPLC analysis of their N‐(3,5‐dinitrobenzoyl)prolyl derivatives 17 is presented.

Construction of an expression system for engineering of the lantibiotic Pep5.

Pep5 is a lanthionine-containing antimicrobial peptide which is produced by Staphylococcus epidermidis 5. Its structural gene, pepA, is located on the 20-kb plasmid pED503. A 6.2-kb fragment of pED503 containing pepA, the immunity gene pepI, and 5.4 kb of downstream sequence was able to direct biosynthesis of biologically active Pep5 in a nonproducing variant of the producer strain which is devoid of pED503. In addition to producing wild-type Pep5 with a molecular mass of 3,488 Da, the clone produced a peptide with an eightfold-lower bactericidal activity and a mass of 3,506 Da, indicative of incomplete dehydration of one hydroxyamino acid. For construction of the expression system, this 6.2-kb fragment was cut into a 1.39-kb fragment containing pepA and pepI and a 4.8-kb fragment covering the remaining downstream region. This 4.8-kb fragment was directly cloned into an Escherichia coli-Staphylococcus shuttle vector, yielding a new plasmid (pGB9) into which mutated pepA genes generated on the 1.39-kb fragment can be reinserted to yield a functional Pep5 biosynthesis gene cluster. To test the expression system, two mutants were constructed. Lys-18-Pro Pep5 was produced in its dehydrated form and a partially hydrated form in amounts comparable to those of the wild-type peptide. In contrast, only small amounts of Phe-23-Asp Pep5 were excreted, indicating that some residues in the propeptide part of the prelantibiotic may be crucial for certain steps in the biosynthetic pathway of lantibiotics.

Combination of high-performance anion-exchange chromatography and electrospray mass spectrometry for analysis of the in vitro O-glycosylated mucin motif peptide.

Reversed-phase high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection were developed for the study of products obtained from the in vitro O-glycosylation of a mucin motif peptide, TTSAPTTS, the most representative sequence encoded by the human gene MUC5C. After incubation of the peptide, which is rich in clustered hydroxyamino acids, by both human colonic and gastric microsomal homogenates, the glycosylated products were separated by HPLC and HPAEC and analysed by electrospray mass spectrometry (ES-MS). The combination of HPAEC and ES-MS was the approach used for evaluating the differences between the polypeptide N-acetylgalactosaminyltransferase activity in different digestive tissues.

Synthesis of β-galactosyl-(hydroxy amino acid) derivatives using β-galactosidase activity ofAchatina achatina digestive juice

The transglycosylation activity of β-galactosidase fromAchatina achatina digestive juice was tested for glycosylating protected hydroxy amino acids. Attractive yields of β-galactosyl-(Z-Ser-OMe) (35%) and β-galactosyl-(Z-Hyp-OMe) (28%) could be obtained using lactose as glycosyl donor and the corresponding amino acid methyl esters N-protected by a benzyloxycarbonyl group (Z) as glycosyl acceptors.

P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP‐dependent protein kinase

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.

Simple and Versatile Synthesis of Optically Active 1,2‐Amino Alcohols by Grignard Addition to N,O‐Dibenzylglyceraldimine and ‐lactaldimine

In many cases with high threo selectivity, Grignard additions can be performed on simple, nonactivated, and even enolizable imine derivatives of optically active aldehydes, as the reactions with 1 and 2 show. This should open a new route to aminohydroxy acids that supplements the familiar one starting from α-amino acids/aldehydes.

Identification and Characterization of O-Biotinylated Hydroxy Amino Acid Residues in Peptides

Recent findings have indicated that certain hydroxyl groups of peptides have enhanced intrinsic chemical reactivity and can be O-acylated by N-hydroxysuccinimide esters of biotin. Analytical procedures are described for the identification of O-acylated derivatives of seryl, threonyl, and tyrosyl residues after reaction with the N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid. The identification and chemical characterization of O-biotinylated residues included amino acid analysis, peptide sequence determination, and mass spectrometric analysis. The application of these analytical procedures provided unequivocal identification of O-biotinylated residues for a number of peptides investigated. In a separate study, we also show that O-biotinylated amino-terminal seryl residues occurring after proteolysis of peptides or proteins can rapidly undergo an O → N acyl shift resulting in blockage of sequence analysis by the Edman degradation procedure.

ESR spectral study of free radical formation during the pyrolysis of hydroxyamino acids

Relatively stable free radicals, formed by pyrolysis of dl-serine, dl-threonine and dl-tyrosine over the temperature range 200-600°C, during reaction periods lasting for between 2 and 180 min, were identified by Electron Spin Resonance ESR spectroscopy. The ESR spectra, which showed broad singlets in all examined cases, suggest that the free radicals were different types of polycondensation species. The concentrations of free radicals formed, measured as the intensities of absorption of the spectral lines, depended on the applied temperatures, the duration of pyrolysis and the structure of the examined amino acids.

IL-2-induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression.

We have recently isolated the cDNA for a unique human 97-kDa kinase, TTK, by expression screening of a cDNA expression library using anti-phosphotyrosine antibodies. When expressed in Escherichia coli, TTK can phosphorylate serine, threonine, and tyrosine residues. Thus TTK appears to belong to a newly described family of kinases able to phosphorylate all three hydroxy amino acids. This family of multispecific kinases includes several other kinases involved in cell cycle progression. In support of a possible role in regulating cell cycle progression, TTK message is readily detected in rapidly proliferating tissues in vivo including testes, thymus, bone marrow, and many malignant tumors, but not in benign tissues with a low proliferative rate in vivo. To determine the effect of cell activation and cell cycle progression on TTK expression, we measured TTK mRNA and protein levels as well as kinase activity in freshly isolated T cells or IL-2-expanded T cell blasts activated to proliferate by the addition of a variety of mitogens. TTK mRNA levels, protein levels, and kinase activity were greatly enhanced when either freshly isolated PBL or T cell blasts were activated by cross-linking the TCR complex by mitogenic lectins or by bypassing the TCR with phorbol esters and cation ionophores. Incubation with IL-2 increased TTK expression in PBL blasts, which proliferate in response to IL-2, but not in fresh PBL, which do not proliferate in response to IL-2. TTK expression was blocked by either cyclosporin A or FK520, which inhibit IL-2 production and could be recovered by the addition of exogenous IL-2. Furthermore, TTK expression was prevented by incubation of the cells with rapamycin, which blocks IL-2 signaling. Thus, TTK expression in T cells appears to be a consequence of IL2-induced cell proliferation. Agonist-induced TTK expression was a delayed event occurring 12 to 24 h after activation of PBL blasts and 48 to 72 h after activation of fresh PBL. TTK protein and mRNA expression increased in both fresh PBL and T cell blasts concurrently with passage of cells through S phase as indicated by [3H]TdR incorporation and cell cycle analysis of propidium iodide-stained cells. TTK mRNA and protein levels reached a maximum as cells entered the G2 phase of the cell cycle. These results were confirmed by cell cycle blockade studies with aphidicolin and nocodazole wherein TTK protein levels are not detected in cells in G1 and are readily detectable in cells in the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the aryl groups as the carboxyl synthon. Application to the synthesis of some natural products containing hydroxyamino acid functions

Abstract

Sulfate esters of hydroxy amino acids as stereospecific glutamate receptor agonists

Enantiomerically pure sulfate esters of the hydroxy amino acids homserine, hydroxyproline and 4-hydroxypipecolic acid were synthesized and tested on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N- methyl- D-aspartate (NMDA) receptors present in the mice cortical wedge preparation and on NMDA receptors present in the myenteric plexus of the guinea pig with the aim of finding new possible ligands (either agonists) for excitory amino acid receptors. The linear and flexible compound S-homoserine sulfate caused a depolarization of both AMPA and NMDA receptors. In the cortex its agonist action had an EC 50 of 150 μM for NMDA and 300 μM for AMPA receptors and in the myenteric plexus its EC 50 was 600 μM. The stereoisomer R-homoserine sulfate did not depolarize the cortical wedges and failed to cause ileal contraction up to a concentration of 500 μM. Among the four possible stereoisomers of 4-hydroxyproline sulfate, which are rigid structures and may be regarded as cyclization forms of homoserine sulfate, t- S-hydroxyproline sulfate was a selective AMPA receptor agonist with an EC 50 of 70 μM in the cortex. The other three isomers were not active as agonists up to 500 μM and none of them had antagonist activity. Finally, t-4- rmhydroxy- S-pipecolic acid-4-sulfate, a superior homologue of t- S-hydroxyproline sulfate, was found to be one of the most potent and selective NMDA receptor agonists so far described with an EC 50 of 0.7 μM in the cortex and 250 μM in the myenteric plexus. The cis-stereoisomer was significantly less potent (EC 50 75 μM in the cortex and no activity up to 500 μM in the myenteric plexus). In conclusion, S-homoserine sulfate, t- S-hydroxyproline sulfate and t-4-hydroxy- S-pipecolic acid-4-sulfate are natural compounds able to interact agonists in a stereospecific and selective manner with ionotropic glutamate receptors.

A New Concise Approach to the Enantioselective Synthesis of the Hydroxyamino Acid Moiety of AI-77-B

The hydroxyamino acid moiety of AI-77-B, pyrrolidin-2-one derivative, has been prepared from D-ribose in an optically pure form via stereoselective alkylation of N-acylpyrrolidinium ion intermediates as the key step

Biogenic Amino Acids and their Metal Salts as Catalysts of Caramelization

AbstractBasic amino acids and one neutral aminohydroxy acid ‐ serine ‐, all at the concentration of 5%(w/w), are the most suitable as catalysts of caramelization. Among basic amino acids asparagine is the superior catalyst. Although caramels obtained with serine as the catalyst showed higher tinctorial strength they do not fully dissolve in alcohol, beer, hydrochloric acid and aq. citric acid. The application of lithium, sodium, and potassium salts of amino acids under study sometimes give an increase of tinctorial strength of caramels which, however, form precipitate in the citric acid test. The choice between particular salts as catalysts can be dependent on requested aroma of the caramel.

ChemInform Abstract: A Simple Total Synthesis of Naturally Occurring Hydroxy Amino Acids by Enzymatic Kinetic Resolution.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Yeast MCK1 protein kinase autophosphorylates at tyrosine and serine but phosphorylates exogenous substrates at serine and threonine.

The product of the Saccharomyces cerevisiae MCK1 gene is a protein kinase that phosphorylates poly (Glu,Tyr) in vitro and is itself phosphorylated at both tyrosine and serine in vivo. To characterize the substrate specificity of Mck1, the enzyme was purified to apparent homogeneity from the soluble fraction of yeast cell extracts by ammonium sulfate precipitation, followed by ion exchange chromatography (Q- and S-Sepharose), dye-ligand affinity chromatography (Orange A-agarose), adsorption chromatography (hydroxylapatite), and ion exchange fast protein liquid chromatography (Mono-S). In the absence of an exogenous substrate, purified Mck1 was able to autophosphorylate on tyrosine and serine. A catalytically inactive mutant (K68R in conserved kinase domain II) expressed in an mck1 delta strain did not contain detectable phosphotyrosine, confirming that the tyrosine phosphorylation observed in vivo is due to autophosphorylation, but did contain phosphoserine, suggesting that Mck1 is a target for other cellular protein kinases. Purified Mck1 phosphorylated a variety of proteins in heat-inactivated yeast extracts, primarily on serine (and threonine). The purified enzyme also used a number of mammalian proteins as phosphoacceptors, including myelin basic protein (MBP), microtubule-associated protein 2 (MAP-2), and tau protein. All of these substrates were phosphorylated on either serine or threonine (or both). Mck1 isolated from yeast extracts by immunoprecipitation with an anti-Mck1 antibody directed against its C terminus also phosphorylated MBP at serine. In the same immune complex kinase assay, the K68R mutant did not detectably phosphorylate MBP, indicating that the serine-specific phosphotransferase activity of Mck1 is intrinsic and not due to contamination by an associated kinase. These findings demonstrate that Mck1 is a member of a novel class of protein kinases that displays the ability to phosphorylate all three hydroxyamino acids in proteins.

A facile synthesis of N-protected statine and analogs via a lipase-catalyzed kinetic resolution

A new synthesis of N-protected statine 4a and its analogue 4b is presented. cis-(±)-Butyric acid 2-isobutyl-5-oxopyrrolidin-3-yl ester (10a) and cis-(±)-butyric acid 2-benzyl-5-oxopyrrolidin-3-yl ester (10b) were prepared from methyl (E)-d-chloro-3-methoxybut-2-enoate (5) and subsequently resolved via a kinetic resolution catalyzed by Candida Cylindracea lipase. The desired enantiomers were isolated in excellent enantiomeric excess and good yield. These products were then converted to the N-BOC-protected hydroxy amino acids 4a and 4b

ChemInform Abstract: Chiral Synthons for 2‐Amino Alcohols. Facile Preparation of Optically Active Amino Hydroxy Acids of Biological Interest.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.