Enzymatic synthesis of polyprenol monophosphate mannose in insects.

The microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 degrees C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an alpha-saturated polyprenol phosphate similar to dolichol monophosphate.     

Enzymatic synthesis and surface active properties of novel hemifluorinated mannose esters

The lipase-catalysed esterification of sugars with hemifluorinated acid derivatives is reported for the first time. A series of mannose modified derivatives having fluorinated chains with different length have been prepared accordingly in moderate yield. A preliminary evaluation of the surface active properties of these hemifluorinated mannose esters revealed their ability to reduce the surface tension of water much more efficiently than their aliphatic counterparts.     

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose.

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to yeast mannan-protein complexes

The radioactive products derived from transfer of [¹⁴C]mannose residues from GDP-[¹⁴C]mannose to endogenous acceptors of a Hansenula holstii particulate enzyme preparation have been solubilized by Pronase digestion. From this soluble mixture, glycopeptides containing [¹⁴C]mannose have been purified and have been shown by β-elimination-reduction experiments to contain radioactive mannose and oligosaccharides of mannose linked to serine and threonine residues. Radioactive macromolecular complexes of mannan-protein were extracted from the particulate enzyme fraction with hot, neutral citrate buffer. These components contained variable quantities of protein, mannose, and phosphate. The more neutral components were reduced in size by Pronase digestion and yielded glycopeptides similar to those obtained by direct Pronase digestion of the particulate fraction.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to dolichol phosphate in yeast (Hansenula holstii) A possible step in mannan synthesis

A particulate enzyme fraction isolated from yeast (Hansenula holstii) catalyzes the transfer of mannose from GDPmannose to endogenous lipid acceptors. Kinetic studies are presented which suggest that one of the mannolipids is a precursor to cell wall mannan. The solubility and chromatographic properties, the stability to mild alkali, and the release of mannose by mild acid hydrolysis are characteristic of polyisoprenyl phosphoryl mannose. Addition of dolichol phosphate to the enzyme system stimulates the synthesis of a mannolipid with properties similar to that synthesized from endogenous lipid. That the exogenous dolichol phosphate was acting as a mannosyl acceptor was demonstrated by showing that dolichol [³²P]phosphate was converted to dolichol [³²P]phosphate mannose.     

Enzymatic dephosphorylation of retinyl monophosphate by rat liver

Rat liver has been shown to contain an enzyme that catalyzes the dephosphorylation of retinyl monophosphate. This activity was extracted with 0.1 M Tris buffer (pH 7.5). Maximum reaction rate was observed at a pH range of 7.0-7.5. It did not require metal ions for activity and was sensitive to fluoride ion. The retinyl monophosphate phosphatase activity was proportional to time and protein and substrate concentration. Triton X-100 (range of 0.05-0.10%) increased the activity 100%, whereas other detergents (Tween 80, cholate, and deoxycholate) did not activate the enzyme. A number of phosphorylated compounds tested as inhibitors of retinyl monophosphatase activity, such as glucose 6-phosphate (20 mM), glycerophosphate (20 mM), phosphatidic acid (8 mM), and dolichyl phosphate (3 mM), did not compete with retinyl monophosphate as substrate. However, at 20 mM concentration, ATP, ADP, 5'-AMP, and pyrophosphate were inhibitors of the enzyme. It is not possible at present to give further details about the specificity of the phosphatase activity. The enzyme described could play a regulatory role in retinol-mediated glycosylations, by altering the endogenous level of retinyl monophosphate.     

The synthesis of mannose 1-phosphate in brain

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.     

Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementationgroup display a distinctive profile of resistance to a varietyof toxic lectins. In addition, they accumulate cis--unsaturatedpolyprenol and use mainly polyprenol rather than dolichol tosynthesize the glycosylated lipids used in asparagine-linkedglycosylation of proteins. The primary defect in these cellsis thought to result from a deficiency in polyprenol reductaseactivity. Three new mutants were isolated and determined tohave qualitatively, although not quantitatively, similar lectinresistance profiles to Lec9 cells. Two of these mutants (Abr^Rand Ric^R) also contained polyprenol rather than dolichol. Thelectin resistance profile of an independent mutant which accumulatespolyprenol, F2A8, was also found to be qualitatively similarto the Lec9 pattern. The relationship among these mutants wasanalysed in more detail by construction of cell—cell hybrids.Lectin resistance profiles of the hybrids demonstrated thatAbr^R, Ric^R and F2A8 fell into the Lec9 complementation group.Analysis of prenols in the hybrids also showed that F2A8 wasa member of the Lec9 group. Surprisingly, a significant fractionof the prenols found in Lec9 Parent hybrids was polyprenol(up to 30% of the neutral fraction), whereas the prenols foundin Parent Parent hybrids were nearly exclusively dolichol(97% of the neutral lipid fraction). Therefore, reduction ofpolyprenol to dolichol appears to be a rate-limiting step inthe synthesis of dolichol since hybrids with differing numbersof wild-type alleles can be biochemically distinguished. CHO cells dolichol lectins mutants polyprenol reductase     

Enzymatic synthesis of chloro-L-tryptophans

4-Chloro-L-tryptophan (1a), 5-chloro-L-tryptophan (1b), 6-chloro-L-tryptophan (1c and 7-chloro-L-tryptophan (1d) were prepared from 4-, 5-, 6- and 7-chloroindoles (2a-d) by reaction with L-serine using tryptophan synthase. The chlorotryptophans were optically pure ( > 99% e.e).     

Enzymatic synthesis of oligosaccharides

As the importance of the oligosaccharide moieties of glycoproteins and glycolipids is being increasingly recognized, efforts to synthesize them are expanding. The number of functional groups of carbohydrate monomers and the variety of configurations that oligomers can adopt is greater than with nucleotides/nucleic acids or amino acids/peptides. By reversing the hydrolytic action of glycosidases and by using highly regiospecific glycosyltransferases, enzymatic oligosaccharide synthesis can be performed.     

Enzymatic synthesis of alkyds

Lipases were used as catalysts in the synthesis of "all-trans" polyester oligomers in organic solvents. Esters of fumaric acid and 1,4-butane diol served as the substrates in the enzyme-catalyzed polytransesterification. No isomerization of the double bond was found under the mild conditions of enzymatic catalysis used by us, as opposed to the extensive isomerization found during chemical polycondensation. The alkoxy leaving group of the ester fumarate was found to be responsible for the rate of transesterification. Low (M(w) approximately 600-800) and high (M(w) = 1250) molecular weight alkyds were synthesized depending on whether tetrahedrofuran or acetonitrile, respectively, was used as the solvent.     

Enzymatic synthesis of L-cysteine

O-Acetylserine sulfhydrase in the form of a crude extract from Salmonella typhimurium LT2 was used for the production of L-cysteine from L-O-acetylserine and sodium hydrosulfide at pH 7.0 and 25 degrees C. The two substrates have quite different pH stability relationships. O-Acetylserine readily rearranges to N-acetylserine and the rate of this O --> N acyl transfer reaction increases at higher pH, temperature, and concentration of O-acetylserine. On the other hand, sodium hydrosulfide is more soluble at a higher pH. A stirred-tank bioreactor with a continuous substrate feed was employed to overcome this problem. The O-acetylserine feed was stored at its saturation level (2.05M) at pH 5.0, and the sodium hydrosulfide feed was dissolved at 2.05-2.3M without pH adjustment (pH >/= 11.5). Both substrates were simultaneously introduced into the bioreactor. The performance of the bioreactor was optimized by employing an automatic feedback control system to regulate the concentration of O-acetylserine in the bioreactor. This feedback control system was based on the fact that as the bioconversion proceeds, protons are produced along with cysteine. A pH controller thus detected the decrease in pH and activated the substrate pumps. After mixing in the bioreactor, these two substrate solutions behaved as a base due to the high alkalinity of sodium hydrosulfide. Thus, substrate infusion started when the pH was lower than the set point, i.e., the reaction pH, and stopped when the pH was raised higher than the set point. The amount of substrate introduced was determined by the alkalinity of the mixture of the two substrates, which in turn was controlled by the concentration of sodium hydrosulfide. After optimizing the sodium hydrosulfide concentration and the substrate feed rate, the bioconversion gave a productivity of 3.6 g L-cysteine/h/g dry cell weight S. typhimurium, an L-cysteine titer of 83 g/L and a molar yield based on O-acetylserine of 94%.