Stimulation of ethylene production by hydroxamic acid, in particular, by benzohydroxamic acid

The effects on the ethylene production of known inhibitors ofa cyanide-insensitive, alternative respiration in plants wereinvestigated using cotyledonary segments of cocklebur (Xanthiumpennsylvanicum Wallr.) seeds. Benzohydroxamic acid (BHAM) at3 mM stimulated ethylene production 4- to 8-fold over the control,but respiration of the segments was hardly affected at thatconcentration. The stimulatory effects of 3-chlorobenzohydroxamicacid (CLAM) and salicylhydroxamic acid were far smaller thanthat of BHAM. BHAM at 3 mM also markedly stimulated the ethyleneformation in the epicotyl or hypocotyl sections of etiolatedpea (Pisum sativum L.) and mung bean (Vigna radiata [L.] Wilczek)seedlings. Moreover, 3 mM BHAM further promoted the increasedethylene formation which was caused by administration of 1-aminocyclopro-pane-1-carboxylicacid (ACC) to the cotyledonary segments. The promoting effectsby BHAM and CLAM were also found in the conversion of ACC intoethylene in pea stem homogenates. (Received July 26, 1980; )     

Stimulation of ethylene production by hydroxamic acid, in particular, by benzohydroxamic acid

The effects on the ethylene production of known inhibitors ofa cyanide-insensitive, alternative respiration in plants wereinvestigated using cotyledonary segments of cocklebur (Xanthiumpennsylvanicum Wallr.) seeds. Benzohydroxamic acid (BHAM) at3 mM stimulated ethylene production 4- to 8-fold over the control,but respiration of the segments was hardly affected at thatconcentration. The stimulatory effects of 3-chlorobenzohydroxamicacid (CLAM) and salicylhydroxamic acid were far smaller thanthat of BHAM. BHAM at 3 mM also markedly stimulated the ethyleneformation in the epicotyl or hypocotyl sections of etiolatedpea (Pisum sativum L.) and mung bean (Vigna radiata [L.] Wilczek)seedlings. Moreover, 3 mM BHAM further promoted the increasedethylene formation which was caused by administration of 1-aminocyclopro-pane-1-carboxylicacid (ACC) to the cotyledonary segments. The promoting effectsby BHAM and CLAM were also found in the conversion of ACC intoethylene in pea stem homogenates. (Received July 26, 1980; )     

Functionalized cyclodextrins as holoenzyme mimics of thiamine-dependent enzymes

Mimics of vitamin B₁-dependent enzymes have been prepared by covalently linking thiazolium salts to the primary side of β-cyclodextrin. Although these compounds were found to catalyze the benzoin condensation, they were generally less effective turnover catalysts than the analogous simple thiazolium salts lacking the artificial binding site. An improvement in the design of the sugar derivative which prevented competitive binding of the thiazolium moiety to the cyclodextrin cavity did, however, lead to a superior catalyst of this transformation. In catalytic processes which required only one molecule of substrate to react with the thiazolium salt, rate accelerations, saturation kinetics, and substrate selectivity were observed. Thus, all the cyclodextrin thiazolium salts speeded the rate of tritium exchange from suitably labeled aromatic aldehydes more than did simple thiazolium salts. In addition, rate enhancements of up to ca 40-fold were observed for the thiazolium-catalyzed oxidation of tert-butylbenzaldehyde by ferricyanide compared with a thiazolium salt lacking the cyclodextrin binding group.     

Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes

Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy:

Chronic thiamine deprivation in the rat leads to selective neuropathological damage in brainstem structures whereas treatment with the central thiamine antagonist, pyrithiamine, results in more widespread damage. In order to further elucidate the neurochemical mechanisms responsible for this selective damage, the thiamine-dependent enzyme complex pyruvate dehydrogenase (PDHC) was measured in 10 brain structures in the rat during progression of thiamine deficiency produced by chronic deprivation or by pyrithiamine treatment. Feeding of a thiamine-deficient diet to adult rats resulted in 5–7 weeks in ataxia and loss of righting reflex accompanied by decreased blood transketolase activities. PDHC activities were selectively decreased by 15–30% in midbrain and pons (lateral vestibular nucleus). Thiamine treatment of symptomatic rats led to reversal of neurological signs and to concomitant reductions of the cerebral PDHC abnormalities. Daily pyrithiamine treatment led within 3 weeks to loss of righting reflex and convulsions and to decreased blood transketolase of a comparable magnitude to that observed in chronic thiamine-deprived rats. No significant regional alterations of PDHC, however, were observed in pyrithiamine-treated rats.     

Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study

Meprins are complex and highly glycosylated multi-domain enzymes that require post-translational modifications to reach full activity. Meprins are metalloproteases of the astacin family characterized by a conserved zinc-binding motif (HExxHxxGFxHExxRxDR). Human meprin-α and -β protease subunits are 55% identical at the amino acid level, however the substrate and peptide bond specificities vary markedly. Current work focuses on the critical amino acid residues in the non-primed subsites of human meprins-α and -β involved in inhibitor/ligand binding. To compare the molecular events underlying ligand affinity, homology modeling of the protease domain of humep-α and -β based on the astacin crystal structure followed by energy minimization and molecular dynamics simulation of fully solvated proteases with inhibitor Pro-Leu-Gly-hydroxamate in S subsites were performed. The solvent accessible surface area curve shows a decrease in solvent accessibility values at specific residues upon inhibitor binding. The potential energy, total energy, H-bond interactions, root mean square deviation and root mean square fluctuation plot reflect the subtle differences in the S subsite of the enzymes which interact with different residues at P site of the inhibitor.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Comparative aspects of plasma membrane-bound enzymes in frog and rat liver

• 1.1. Plasma membranes have been isolated from frog (Rana esculenta) liver.
• 2.2. The average yield was 0.194 mg protein/g wet liver.
• 3.3. The activities of plasma membrane-bound enzymes (Na⁺-K⁺-ATPase and 5'-nucleotidase as well as of (Mg²⁺)-ATPase have been determined in the liver homogenate and in isolated plasma membranes.
• 4.4. (Na⁺-K⁺-ATPase, 5'-nucleotidase and (Mg²⁺)-ATPase activities of frog liver are compared with the same enzymatic activities observed in rat liver.

     

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

Chemical engineering aspects of submerged production of citric acid

We determined the total mass balance of production of citric acid byAspergillus niger on the laboratory scale and derived a stoichiometric equation for optimum conditions. At the aeration intensity of 4.10^-3 m³/min, 2/3 of introduced carbon was found to be converted to citric acid. Other parameters were: rate of absorptionNa, 15 –17 mol^-3h^-1 and oxygen utilization equal to 42 % of sugar mass. The critical concentration of dissolved oxygen,i.e. 9.5 % of saturation level, was not exceeded.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: (i) equally reduced in magnitude in vulnerable and non-vulnerable brain structures, (ii) unchanged following reversal of neurological abnormalities by thiamine administration.     

Inhibition of urease activity by hydroxamic acid derivatives of amino acids.

Hydroxamic acids have been reported to be potent and specific inhibitors of urease (EC 3.5.1.5) activity of plant and bacterial origin. The present investigation was performed on the inhibitory effect of hydroxamic acid derivatives of naturally occurring amino acids on the urease activity of the Jack Bean and the alimentary tracts of rats. Methionine-hydroxamic acid was the most powerful inhibitor (I50=3.9 X 10(-6) M) among nineteen alpha-aminoacyl hydroxamic acids. Phenylalanine-, serine-, alanine-, glycine-, histidine-, threonine-, leucine-, and arginine-hydroxamic acids followed, in order of decreasing inhibitory power. The inhibition proceeded with time at a comparable rate to fatty acyl hydroxamic acid inhibition. The I50 values of alpha-aminoacyl hydroxamic acids were found to be almost equal to those of the corresponding fatty acyl hydroxamic acids. This fact shows that the alpha-amino group did not affect inhibitory power. However, aspartic-beta-, lysine-, and glutamic-gamma-hydroxamic acids, in descending order, were much less inhibitory, probably due to the presence of a carboxyl or omega-amino group. Furthermore, the pH optimum of the inhibition shifted to lower pH in the presence of a carboxyl group, and to a higher pH in e presence of an amino group. The results suggest that the dissociation of an acidic or a basic group reduces the inhibitory power of hydroxamic acid. Hydroxamic acid inhibits urease activity with strict specificity, excpet for aspartic-beta-hydroxamic acid, which inhibited asparaginase competitively. Hydroxamic acid derivatives of amino acids inhibited not only the urease activity of the Jack Bean, but also that of the caecum and ileum parts of the rat intestine.     

Inhibition of ATPase from chloroplasts by a hydroxamic acid from the gramineae

-DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits     

Comparative inactivation of yeast fatty acid synthetase component enzymes by 100 atmospheres of oxygen.

Yeast fatty acid synthetase at 4 degrees C was stable during 1- and 2-h exposures to oxygen at 100 atm, but was 48% and 90% inactivated after 20 h and 40 h, respectively, with fatty acid synthesis measured by both radioactive and optical assays. Incubation with dithiothreitol did not restore activity. Component enzyme activities were compared before and after 40 h in 100 atm of oxygen. Ketoacyl reductase activity was most reduced followed by ketoacyl synthetase and then acetyl transferase while malonyl transferase, enoyl reductase and palmitoyl transferase were not significantly inactivated.     

Stimulatory effect of ferulic acid on the production of extracellular xylanolytic enzymes by Aspergillus kawachii

Production of extracellular beta-1,4-xylanase, alpha-L-arabinofuranosidase, feruloyl esterase, and acetyl xylan esterase from Aspergillus kawachii was higher in a culture supplemented with ferulic acid than in a counterpart. Culture supernatant grown on oat spelt xylan supplemented with ferulic acid exhibited an increase in ferulic acid-releasing activity from insoluble arabinoxylan relative as compared to that from the ferulic acid-free culture.     

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.     

Effects of maternal thiamine deficiency on the development of thiamine-dependent enzymes in regions of the rat brain

Previous studies suggest that developing rat brain is susceptible to reduced thiamine intake. In order to assess the metabolic basis for this susceptibility, activities of three thiamine-dependent enzymes (pyruvate dehydrogenase complex, -ketoglutarate dehydrogenase and transketolase) were measured in homogenates of brain tissue from the offspring of thiamine-deficient mothers. Control groups of animals were pair-fed to equal food consumption with the thiamine-deficient animals. The study revealed region-selective delays in the establishment of adult activities of thiamine-dependent enzymes as a result of maternal thiamine deficiency. Pyruvate dehydrogenase complex activities in cerebral cortex were significantly reduced (by 20% P < 0.05); -ketoglutarate dehydrogenase activities were also reduced in cerebral cortex (by 30% P < 0.05). In the case of transketolase, enzyme activities were significantly reduced in cerebral cortex, cerebellum and brainstem. Following thiamine replenishment, defective enzyme activities were restored to normal in all cases. However, since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of thiamine-dependent enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.