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T Shimakata

Publications and source records attributed to T Shimakata.

At least 19 recordsLinked to original sources
PubMed2000

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii.

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.

Adenosine Triphosphate↗
PubMed1986

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii.

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.

Actinomycetaceae↗
PubMed1985

Mass-spectrometric identification of trehalose 6-monomycolate synthesized by the cell-free system of Bacterionema matruchotii.

The fluffy layer fraction prepared from Bacterionema matruchotii was found to possess high activity for the biosynthesis of mycolic acids which were bound to an unknown compound by an alkali-labile linkage [T. Shimakata, M. Iwaki, and T. Kusaka (1984) Arch. Biochem. Biophys. 229, 329-339]. To determine the structure of the mycolate-containing compound, it was purified and analyzed by field desorption (FD) and secondary ion mass spectrometry (SI-MS). When non-labelled palmitic acid was used as a precursor in the in vitro biosynthetic system, the underivatized product had a cationized molecular ion, [M + Na]+, at m/z 843 in FD-MS and a protonated ion, [M + H]+, at m/z 821 in SI-MS, corresponding to the quasimolecular ion of trehalose monomycolate (C32:0). In SI-MS, characteristic fragment ions due to cleavage of glycosidic linkages were clearly detected in addition to the molecular ion. If [1-13C]palmitic acid was the precursor, 2 mass unit increases in both the quasimolecular and fragment ions were observed, indicating that two molecules of palmitate were incorporated into the product. alpha-Trehalose was found in the aqueous phase after saponification of the product. By the electron impact mass spectrometry of the trimethylsilylated product, the mycolate was found to be esterified with an hydroxyl group at position 6 of the trehalose molecule. These results clearly demonstrated that the predominant product synthesized by the fluffy layer fraction with palmitate as substrate was 6-monomycolate (C32:0) of alpha-D-trehalose. Because newly synthesized mycolic acid was mainly in the form of trehalose monomycolate instead of free mycolate or trehalose dimycolate, the role of trehalose in the biosynthesis of mycolic acid is discussed.

Actinomycetaceae↗
PubMed1984

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii.

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-14C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C32:0 mycolic acid by radio-gas-liquid chromatographic (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-14C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C34:0 + C34:1) mycolic acids and the other to (C36:0 + C36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C32:0 mycolic acid synthesized by [1-14C]palmitic acid was pyrolyzed at 300 degrees C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-14C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from 14C-palmitate whereas cerulenin, a specific inhibitor of beta-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-14C]fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.

Actinomycetaceae↗
PubMed1983

The purification and function of acetyl coenzyme A:acyl carrier protein transacylase.

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.

Acetyltransferases↗
PubMed1983

Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves.

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1982

Isolation and function of spinach leaf beta-ketoacyl-[acyl-carrier-protein] synthases.

Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When beta-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates palmitoyl-ACP to beta-ketostearoyl-ACP but has no activity towards decanoyl-ACP. The first protein is designated beta-ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second protein is designated beta-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification of synthase II is described, and its activity is compared to synthase I. Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis.

Journal Article↗
PubMed1982

Fatty Acid Synthetase of Spinacia oleracea Leaves.

The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthetase, beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system.

Journal Article↗
PubMed1981

Purification and characterization of 2-enoyl-CoA reductase of Mycobacterium smegmatis.

2-Enoyl-CoA reductase was purified to homogeneity for the first time from the crude extract of Mycobacterium smegmatis. Its molecular weight was estimated to be 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted as an electron donor for the reduction of 2-enoyl-CoA, while NADPH did not. The Km value for NADH was 21.3 microM. On the other hand, NAD inhibited the reaction for competing against NADH, as the Ki value for NAD was 47 microM. Among the enoyl-CoAs used as substrates, those having C10-C16 were found to be most suitable substrates for the purified reductase in terms of both apparent Km and Vmax values. The enzyme was strongly inhibited, however, when the concentration of the C16-substrate was over 50 microM. The enzyme had almost no activity towards substrates having less than C8. When NAD3H was used as an electron donor to 2-dodecenoyl-CoA in the presence of the purified reductase, only laurate was tritiated as the product. Diacetyl and phenylglyoxal, agents that react specifically with arginine, inactivated the reductase in a time- and concentration-dependent manner during the preincubation. These results suggest that some arginine residues in the reductase protein are involved in the enzyme activity.

Diacetyl↗
PubMed1980

Purification of two forms of enoyl-CoA hydratase from Mycobacterium smegmatis.

Two forms of enoyl-CoA hydratase (hydratases I and II), which are different from each other in substrate specificity, were found in a crude extract of Mycobacterium smegmatis. Hydratase I was more active with crotonyl-CoA as a substrate than with decenoyl-CoA, whereas the reverse was the case for hydratase II. Hydratase I was purified 688-fold to homogeneity with a yield of 14.5% from the crude extract. Its molecular weight was estimated to be 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 30,000 by gel filtration, suggesting that the enzyme is dimeric. Hydratase II was also partially purified. The Vmax of hydratase I decreased progressively with increase in the carbon-chain length of the substrate from 2,488 units/mg for crotonyl-CoA to 154 units/mg for hexadecenoyl-CoA, whereas the Km values for crotonyl-CoA (82 microM), decenoyl-CoA (91 microM), and hexadecenoyl-CoA (105 microM) were similar. Both hydratases were inhibited by acetoacetyl-CoA and pCMS, but not by N-ethylmaleimide or monoiodoacetate.

Enoyl-CoA Hydratase↗