Enzymatic reduction of phenylglyoxal and 2,3-butanedione, two commonly used arginine-modifying reagents, by the ketoacyl reductase domain of fatty acid synthase

Fatty acid synthase catalyzes the reduction of one of the carbonyl groups in phenylglyoxal and 2,3-butanedione using NADPH as the reductant. Selective inactivation of the enoyl reductase, one of the two reductase domains that could catalyze this reduction, did not affect the carbonyl reduction showing that the ketoreductase domain catalyzed the reaction. The apparent Km for the two arginine-specific reagents were lower than that for 3-acetoacetyl-N-acetyl cysteamine, the commonly used model substrate for the ketoreductase activity of the synthase.     

The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K_m of 0.09 mm and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K_m of 0.17 mm and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K_m of 0.67 mm and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α₄β₄ type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K⁺, Rb⁺ and Cs⁺ but not by Na⁺ or Li⁺ and is inhibited by high concentrations of NH₄⁺ and C1^?. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated onlyS-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.     

Isolation and Characterization of Acyl Coenzyme A Carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis, Which Produce Multiple Methyl-Branched Mycocerosic Acids

Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin–Sepharose 4B-CL gel with d-biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 μmol min⁻¹ mg⁻¹, respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K_m values for propionyl-CoA, ATP, Mg²⁺, and NaHCO₃ were 70 μM, 100 μM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K_m was higher and the V_max was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively (S)-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n-fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164     

Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi.

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Biosynthesis of secondary alcohols and ketones from alkanes

Combined gas-liquid chromatography-mass spectrometry of the secondary alcohols and ketones from broccoli (Brassica oleracea) showed that the former consisted of a mixture of nonacosan-14-ol (43%) and nonacosan-15-ol (57%) whereas the latter contained predominantly nonacosan-15-one (92%). Chemical degradation of the secondary alcohols derived from [4-¹⁴C]stearic acid in B. oleracea indicated that the intact C₁₈ chain was incorporated into nonacosan-14-ol and nonacosan-15-ol and that the hydroxyl groups of the positional isomers were introduced into a preformed aliphatic chain. [G-³H]Nonacosane was incorporated into nonacosan-14-ol and nonacosan-15-ol in B. oleracea in a ratio similar to that found in the natural mixture. A randomly tritiated natural mixture of secondary alcohols was incorporated into the ketone fraction by broccoli leaves. Chemical degradation of substrate secondary alcohols and the ketones biologically derived from them demonstrated that nonacosan-15-ol was the preferred secondary alcohol for biological oxidation to the ketone. The incorporation of [G-³H]nonacosane into nonacosanol and nonacosanone by broccoli leaves required O₂ and was inhibited by phenanthroline. The inhibition was reversed by Fe²⁺ suggesting that conversion of nonacosane into the oxygenated derivatives involve a mixed-function oxidase. From these results it is concluded that in B. oleracea nonacosane is hydroxylated to give nonacosan-15-ol and nonacosan-14-ol with subsequent preferential oxidation of the C-15 isomer to give the ketones.