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A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1 H NMR, 13 C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 μM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC 99 2.55 μg/mL whereas compounds 5d and 5j showed activity with MIC 99 18.91 μg/mL and 25.07 μg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC 99 value of 2.55 μg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target., (© 2024 John Wiley & Sons Ltd.)

Summary: Seed vigour is an imperative trait for the direct seeding of rice. Isopropylmalate synthase (IPMS) catalyses the committed step of leucine (Leu) biosynthesis, but its effect on seed vigour remains unclear. In this study, rice OsIPMS1 and OsIPMS2 was cloned, and the roles of OsIPMS1 in seed vigour were mainly investigated. OsIPMS1 and OsIPMS2 catalyse Leu biosynthesis, and Leu feedback inhibits their IPMS activities. Disruption of OsIPMS1 resulted in low seed vigour under various conditions, which might be tightly associated with the reduction of amino acids in germinating seeds. Eleven amino acids that associated with stress tolerance, GA biosynthesis and tricarboxylic acid (TCA) cycle were significantly reduced in osipms1 mutants compared with those in wide type (WT) during seed germination. Transcriptome analysis indicated that a total of 1209 differentially expressed genes (DEGs) were altered in osipms1a mutant compared with WT at the early germination stage, wherein most of the genes were involved in glycolysis/gluconeogenesis, protein processing, pyruvate, carbon, fructose and mannose metabolism. Further analysis confirmed that the regulation of OsIPMS1 in seed vigour involved in starch hydrolysis, glycolytic activity and energy levels in germinating seeds. The effects of seed priming were tightly associated with the mRNA levels of OsIPMS1 in priming seeds. The OsIPMS1 might be used as a biomarker to determine the best stop time‐point of seed priming in rice. This study provides novel insights into the function of OsIPMS1 on seed vigour and should have practical applications in seed priming of rice. [ABSTRACT FROM AUTHOR]

The glyoxylate shunt is an anaplerotic bypass of the traditional Krebs cycle. It plays a prominent role in Mycobacterium tuberculosis virulence, so it can be exploited for the development of antitubercular therapeutics. The shunt involves two enzymes: isocitrate lyase (ICL) and malate synthase (GlcB). The shunt bypasses two steps of the tricarboxylic acid cycle, allowing the incorporation of carbon, and thus, refilling oxaloacetate under carbon-limiting conditions. The targeting of ICL is complicated; however, GlcB, which accommodates the pantothenate tail of acetyl-CoA in the active site, is easier to target. A catalytic Mg2+ unit is located at the bottom of the cavity, and plays a very important role. Recently, the development of effective antituberculosis drugs based on phenyldiketo acids (PDKAs) has been reported. Interestingly, all the crystal structures of GlcB-inhibitor complexes exhibit close contact between the carboxylate of Asp633 and the face of the aromatic ring of the inhibitor. Remarkably, the replacement of the phenyl ring in PDKA by aliphatic moieties yields inactive inhibitors, suggesting that the aromatic moiety is crucial for inhibition. However, the aromatic ring of PDKA is not electron-deficient, and consequently, the anion-π interaction is expected to be very weak (dominated only by polarization effects). Herein, through a combination analysis of the recent X-ray structures of GlcB-PDKA complexes retrieved from the protein data bank (PDB) and computational ab initio studies (RI-MP2/def2-TZVP level of theory), we demonstrate the prominent role of the Mg2+ ion in the active site, which promotes long-range enhancement of the anion-π interaction. [ABSTRACT FROM AUTHOR]

Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism., A novel malic enzyme, Sg ME1, was identified from the Al-tolerant genotype TPRC2001-1 after 72 h Al exposure by two-dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean ( Phaseolus vulgaris) hairy roots., Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001-1, as indicated by 5.8-fold higher root malate concentrations and approximately two-fold higher Al concentrations in roots and root symplasts of TPRC2001-1 than those of the Al-sensitive genotype Fine-stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001-1. Gene and protein expression of Sg ME1 was only enhanced in TPRC2001-1 after 72 h Al exposure. Overexpressing Sg ME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation., These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al-enhanced malate synthesis, functionally controlled by Sg ME1. [ABSTRACT FROM AUTHOR]

• Root exudates play a key role during the presymbiotic growth phase and have been shown to stimulate hyphal branching and the catabolic metabolism of arbuscular mycorrhizal (AM) fungal spores. • Here, the effect of root exudates on presymbiotic growth, uptake of exogenous carbon and transcript levels for genes putatively involved in the carbon metabolism of germinating spores were determined. • Crude root exudates led to a slight acceleration of spore germination, increased germ tube branching and stimulated uptake and catabolic metabolism of acetate, and to a greater extent of glucose, but had no effect on gene expression. By contrast, partially purified root exudates increased the transcript levels of acyl-CoA dehydrogenase (ß-oxidation of fatty acids to acetyl-CoA), malate synthase (glyoxylate cycle) and glutamine-fructose-6-phosphate aminotransferase (chitin biosynthesis), but did not differ from crude root exudates in their effect on substrate uptake and respiration. The expression of glycogen synthase (glycogen biosynthesis), glucose-6-phosphate dehydrogenase (pentose phosphate pathway) and neutral trehalase (hydrolysis of trehalose) were only marginally or not affected by root exudates. • Root exudates have an effect on both membrane activity and gene expression and the results are discussed in relation to the catabolic and anabolic metabolism of spores during presymbiotic growth. [ABSTRACT FROM AUTHOR]

Enzymes of the glyoxylate shunt have been implicated as virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. Malate synthase has thus emerged as a promising target for design of anti-microbial agents. For this effort, it is essential to have reliable models for enzyme:substrate complexes. A 2.7 Å resolution crystal structure for M. tuberculosis malate synthase in the ternary complex with magnesium, malate, and coenzyme A has been previously described. However, some unusual aspects of malate and Mg++ binding prompted an independent determination of the structure at 2.3 Å resolution, in the presence of saturating concentrations of malate. The electron density map of the complex reveals the position and conformation of coenzyme A to be unchanged from that found in the previous study. However, the coordination of Mg++ and orientation of bound malate within the active site are different. The revised position of bound malate is consistent with a reaction mechanism that does not require reorientation of the electrophilic substrate during the catalytic cycle, while the revised Mg++ coordination is octahedral, as expected. The results should be useful in the design of malate synthase inhibitors. [ABSTRACT FROM AUTHOR]

Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success. [ABSTRACT FROM AUTHOR]

Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl-coenzyme A (acetyl-CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 Å-resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl-CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl-CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl-CoA in the rate-limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine-sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X-ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents. [ABSTRACT FROM AUTHOR]

Summary • The ectomycorrhiza is a symbiotic organ formed between a filamentous fungus and a plant root, mainly tree roots. Root colonization involves significant shifts in gene expression resulting in metabolic and structural changes in the fungus, including growth toward the plant root, penetration and establishment of the symbiotic organ. • The preinfection stage of the association is crucial as changes that occur throughout mycorrhiza formation are set in motion. Using an in vitro system for identifying preinfection stage symbiosis-regulated genes from the Laccaria bicolor–Pinus resinosa interaction we have identified a malate synthase from L. bicolor (Lb-MS ). • The glyoxylate pathway, of which malate synthase is an enzyme, acts as a tricarboxylic acid pathway bypass generating four-carbon compounds for biosynthesis. While it is anticipated that malate synthase would be a part of the genetic and metabolic machinery of any fungus, Lb-MS is of interest because it is symbiosis regulated. • Lb-MS is regulated through interaction between the fungus and the host, by glucose and by the presence of other carbon sources in the medium. Its proposed role in the symbiosis is in the utilization of two carbon compounds formed from catabolic processes in early interaction facilitating hyphal net growth. [ABSTRACT FROM AUTHOR]

Structural and cytochemical comparisons were made between three peroxisome types in soybean [Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes. were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations., internal structures, enzyme constitutions and packaging that reflect their metabolic differences. [ABSTRACT FROM AUTHOR]

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic add was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [14C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1 3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide. [ABSTRACT FROM AUTHOR]

Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.

The mechanism of mobilization and utilization of lipids by the developing seedling and the involvement of different tissues in this process was examined in germinating coconut. The endosperm (lipid reserve) and haustorium (spongy tissue developed during germination) were separated from germinating coconuts at intervals of two weeks up to week 22, and assayed for lipase, isocitrate lyase, malate synthase, and catalase, as well as for lipids and sugars. Except for lipase, activities of all the other enzymes were absent in the endosperm during the entire period of germination. However, these enzymes were found to be activated after the tenth week after germination and plateaued at week 16. Induction of these enzymes indicated the operation of the glyoxylate cycle in the haustorial tissue. Glyoxysomes also were observed in the haustorium. The haustorium was shown to absorb triacylglycerol and free fatty acids from endosperm for subsequent conversion to sugars in this tissue. Haustorium therefore is a biochemically active tissue and is the site of operation of the glyoxylate cycle in germinating coconut. [ABSTRACT FROM AUTHOR]