Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Dihydroperoxidation facilitates the conversion of lipids to aldehydic products via alkoxyl radicals

The mechanisms of formation of many aldehydic lipid oxidation products remain unclear, and the involvement of peroxyl radical additions in some cases was recently suggested. Here, the effect of α-tocopherol, a peroxyl radical trap, on the formation of aldehydic phospholipids from a phosphatidylcholine hydroperoxide was studied by electrospray ionization mass spectroscopy (ESI-MS). Based on the observed differential enhancement or suppression of formation of different aldehydes, new pathways are deduced for aldehyde formation via alkoxyl radicals derived from dihydroperoxy derivatives.     

Isolation and nucleotide sequence of the gene encoding phosphoenolpyruvate carboxykinase from Kluyveromyces lactis

The KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK; ATP-dependent) was cloned from the Kluyveromyces lactis genome using a PCR amplicon from Saccharomyces cerevisiae PCK1 gene as a probe. A DNA fragment of about 4·8 kb containing KlPCK1 complemented PEPCK activity of the mutant of S. cerevisiae defective in PEPCK. The KlPCK1 gene has an open reading frame of 1629 bp (543 amino acids). The KlPCK1 nucleotide sequence and deduced amino acid sequence showed 76% and 84% homologies to those of S. cerevisiae PCK1, respectively. Multiple alignment of ATP-dependent PEPCK genes shows highly conserved regions. The nucleotide sequence of KlPCK1 has been submitted to the DDBJ/GenBank/EMBL data bank with Accession Number U88575. © 1998 John Wiley & Sons, Ltd.     

Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein in Saccharomyces cerevisiae

Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr₄₉₂–Thr₅₁₂), corresponding to the β8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu₅₁₃–Lys₅₄₂), corresponding to α-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of α-amylase at non-permissive temperature. © 1998 John Wiley & Sons, Ltd.     

New Si–C bond forming reactions over solid-base catalysts

The reactions of silanes with carbanions generated on the surface of solid bases bring about the nucleophilic substitution at the Si atom to form Si–C bonds. The reaction of alkynes with silanes afforded alkynylsilanes. For example, the reaction of tert-BuCCH or n-BuCCH with Et₂SiH₂ in the presence of KNH₂/Al₂O₃ gave tert-BuCCSiEt₂H and n-BuCCSiEt₂H in a 77% and 67% yield, respectively, at 329 K. Toluene also reacted with Et₂SiH₂ at 329 K to yield benzyldiethylsilane in a 85% yield. The reactions of 1-alkynes with Me₃SiCCH in the presence of KNH₂/Al₂O₃ or KF/Al₂O₃ resulted in a novel type of metathesis reaction between the two alkynes. For example, the reaction of PhCCH with Me₃SiCCH afforded PhCCSiMe₃ and HCCH in a high yield. These new types of base-catalyzed reactions provide new synthetic routes for Si–C bond formation.     

Effects of long-term feeding of marine oils with different positional distribution of eicosapentaenoic and docosahexaenoic acids on lipid metabolism, eicosanoid production, and platelet aggregation in hypercholesterolemic rats

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were distributed mainly in the sn-1,3 positions of seal oil triglyceride and in the sn-2 position of squid oil triglyceride. Seal oil-rich or squid oil-rich fats having constant saturated/monounsaturated/polyunsaturated fatty acid (PUFA) and n−6/n−3 PUFA ratios were fed to exogenously hypercholesterolemic rats for 160 d. The control fat contained linoleic acid as the sole PUFA. Before starting the experimental diets, rats were orally treated with high doses of vitamin D for 4 d to accelerate atherogenesis. The percentage of arachidonic acid in phosphatidylcholine and phosphatidylethanolamine of liver, platelets, and aorta was lower in the marine oil groups than in the control group, seal oil being more effective than squid oil. Maximal platelet aggregation induced by collagen was significantly lower both marine oil groups. Platelet thromboxane (TX) A₂ production induced by collagen or thrombin was markedly reduced by feeding seal or squid oils, the reduction being more pronounced in the seal oil than in the squid oil group. Aortic prostacyclin (PGI₂) production was the same among the three groups. The ratio of the productions of aortic PGI₂ and platelet TXA₂ was significantly higher in the seal oil than in the control group. Although there was no difference in intimal thickness among the three groups, the aortic cholesterol content was significantly lower in the marine oil groups than in the control group. These results showed that the main effects in rats of the different intramolecular distributions of EPA and DHA in dietary fats were on arachidonic acid content in tissue phospholipids and on platelet TXA₂ production.     

Training squirrel monkeys (Saimiri sciureus) to deceive: Acquisition and analysis of behavior toward cooperative and competitive trainers.

Three squirrel monkeys (Saimiri sciureus) learned to reach toward a container that covered food if a cooperative trainer rewarded such reaches by giving the food. A competitive trainer kept any food found, but wrong selections by this trainer were also rewarded. The monkeys initially reached toward the baited container indiscriminately, but gradually and with the aid of color-cued containers, all 3 reliably reached "honestly" and "deceptively" in the presence of the cooperative and competitive trainers, respectively. The monkeys did not appear to take the trainers' knowledge about the location of the food into account, and deception did not occur if food was placed under the normally unbaited container. With additional containers present, monkeys misled the competitive trainer into selecting the unbaited container farthest from the baited one. Although not indicative of mental attribution, the monkeys' behavior suggests awareness of the acquired communicative function of the reaching response. (PsycINFO Database Record (c) 2010 APA, all rights reserved)