Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and α cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in α cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

Genetic control of the arginine pathways in Aspergillus nidulans. Common regulation of anabolism and catabolism

Summary Two regulatory mutants for arginine catabolism isolated as proline suppressors were tested for the synthesis of ornithine transcarbamylase (OTC), the arginine anabolic enzyme. Mutations at one locus, suD, result in the insensitivity of OTC synthesis to effectors responsible for the enzyme level in the wild strain. The common genetic regulation of both catabolic and anabolic pathways of arginine is postulated.     

ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors.     

Cancer cell sensitivity to arginine deprivation in vitro is not determined by endogenous levels of arginine metabolic enzymes

Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg‐deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.     

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

Background  

Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine.     

Effect of N-Methyl Substituents in 2-Methoxycarbonylphenylsulfamides on Kinetics and Mechanism of Formation of (1H)-2,1,3-Benzothiadiazin-4(3H)-One-2,2-Dioxides

The cyclization reactions of N-methyl-N’-(2-methoxycarbonylphenyl)sulfamide (1a), N-methyl-N-(2-methoxycarbonylphenyl)-sulfamide (2a), and 2-methoxycarbonylphenylsulfamide (3a) were studied in aqueous amine buffers (butylamine, ethanolamine, morpholine, glycinamide). The dependences observed between the rate constants and buffer concentrations show that the reactions are subject to base catalysis in all the three cases, the decomposition of the tetrahedral intermediate being rate limiting. The ratio of the relative rate constants of the base catalyzed cyclizations reactions of the three derivatives is 1a: 2a: 3a = 1: 20000: 100. The logarithm of rate constants of the base catalyzed cyclization reactions was plotted against the pK_a values of conjugated acids of the individual amines used as the buffers in the cyclization of compound 1a, and the value of the Brönsted coefficient obtained was about 0.1, which means that the proton transfer from the intermediate to the basic buffer component is thermodynamically favorable. The intermediate is a much weaker base, and the reaction is controlled by diffusion. The slope of an analogous dependence for compound 2a gradually decreases from values near to 0.5 to values near to zero, which means that the intermediate formed from compound 2a (pK_a ≈ 9.3) has a pK_a value comparable with that of the acid buffer component.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis

Key message

The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied.

Abstract

Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant–aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K _m and K _cat values were 7.90 mM and 52.31 s^?1, respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe¹²⁶ is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.     

Subunit organization of Mcm2-7 and the unequal role of active sites in ATP hydrolysis and viability

The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA⁺ ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA⁺ proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA⁺ configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.     

Biotransformation of Methylphenylacetonitriles by Brazilian Marine Fungal Strain Aspergillus sydowii CBMAI 934: Eco-friendly Reactions

This study reports the biotransformation of methylphenylacetonitriles by Brazilian marine filamentous fungus Aspergillus sydowii CBMAI 934 under eco-friendly reaction conditions. The phenylacetonitrile 1, 2-methylphenylacetonitrile 2, 3-methylphenylacetonitrile 3, and 4-methylphenylacetonitrile 4 were quantitatively biotransformed into 2-hydroxyphenylacetic 1a, 2-methylphenylacetic acid 2a, 3-methylphenylacetic acid 3a, and 4-methylphenylacetic acid 4a by enzymatic processes using whole cell as biocatalyst. The marine fungus A. sydowii CBMAI 934 is thus a promising biocatalyst for the preparation of important carboxylic acids under mild conditions (pH 7.5 and 32 °C) from nitrile compounds.     

Prodrugs of fumarate esters for the treatment of psoriasis and multiple sclerosis—a computational approach

Density functional theory (DFT) calculations at B3LYP/6-31 G (d,p) and B3LYP/6-311?+?G(d,p) levels for the substituted pyridine-catalyzed isomerization of monomethyl maleate revealed that isomerization proceeds via four steps, with the rate-limiting step being proton transfer from the substituted pyridinium ion to the C=C double bond in INT1. In addition, it was found that the isomerization rate (maleate to fumarate) is solvent dependent. Polar solvents, such as water, tend to accelerate the isomerization rate, whereas apolar solvents, such as chloroform, act to slow down the reaction. A linear correlation was obtained between the isomerization activation energy and the dielectric constant of the solvent. Furthermore, linearity was achieved when the activation energy was plotted against the pK _a value of the catalyst. Substituted-pyridine derivatives with high pK _a values were able to catalyze isomerization more efficiently than those with low pK _a values. The calculated relative rates for prodrugs 1–6 were: 1 (406.7), 2 (7.6?×?10⁶), 3 (1.0), 4 (20.7), 5 (13.5) and 6 (2.2?×?10³). This result indicates that isomerizations of prodrugs 1 and 3–5 are expected to be slow and that of prodrugs 2 and 6 are expected to be relatively fast. Hence, prodrugs 2 and 3–5 have the potential to be utilized as prodrugs for the slow release of monomethylfumarate in the treatment of psoriasis and multiple sclerosis.

New phenotypic characteristics of three tmm alleles in Arabidopsis thaliana

Key message

Three new tmm mutants were isolated and showed differential phenotypes from tmm - 1 , and TMM overexpression led to abnormal leaf trichomes.

Abstract

TOO MANY MOUTH (TMM) plays a significant role in the stomatal signal transduction pathway, which involves in the regulation of stomatal distribution and patterning. Three mutants with clustered stomata were isolated and identified as new alleles of tmm. tmm-4 mutation included a base transversion from adenine to thymidine in position 1,033 of the TMM coding region and resulted in premature termination of translation at position 345 of TMM. tmm-5 had a base transition from cytosine to thymidine in 244 of TMM and translated 82 amino acids before premature termination. tmm-6 mutation took a base transition from guanine to adenine in 463 of TMM and changed a glycine (Gly) to an arginine (Arg) in position 155 of the protein. tmm-6 had an evident reduction of stomatal clusters and fewer stomata in cluster compared with other tmm alleles, possibly due to decreased level of entry divisions in cells next to two stomata or their precursors. tmm-5 and tmm-6 were hypersensitive to abscisic acid (ABA) in seedling growth and seed germination, while tmm-4 was defective in response to ABA during seed dormancy, suggesting that TMM was involved in ABA signaling transduction. Interestingly, overexpression of TMM resulted in the reduction of leaf trichomes and their branches, and this might reveal a new function of TMM in trichome development.     

Switch-domain mutations in the Saccharomycescerevisiae G protein α-subunit Gpa1p identify a receptor subtype-biased mating defect

The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (α subunit), Ste4p (β) and Ste18p (γ). The switch II region of Gα subunits is involved in several protein-protein interactions and an intrinsic GTPase activity. To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations. The Q323 analog in Gα subunits and Ras is implicated in GTP hydrolysis. Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity. Mutation of residue A59 of Ras and the analogous Gαs residue has had quite different effects. The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in α cells than in a cells. In addition the G321T mutation resulted in an unusual pheromone response phenotype. We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and α-factor receptors.     

Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α_1B-adrenoceptor subtype (selectivity ratios, α_1B/α_1A?=?13, α_1B/α_1D?=?38–39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α₁-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (?)-2, (+)-3, (?)-4–(?)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (?)-1, (?)-3, (+)-6, and (?)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (?)-6 improved in a significant way the α_1B selectivity of the progenitor compound: 4 and 14 time vs. the α_1D subtype and 35 and 77 times vs. the α_1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α₁-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α_1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.