Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast

Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits.     

The anti-inflammatory effects of soluble epoxide hydrolase inhibitors are independent of leukocyte recruitment

Excess leukocyte recruitment to the lung plays a central role in the development or exacerbation of several lung inflammatory diseases including chronic obstructive pulmonary disease. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid reported to have multiple biological functions, including blocking of leukocyte recruitment to inflamed endothelium in cell culture through reduction of adhesion molecule expression. Inhibition of the EET regulatory enzyme, soluble epoxide hydrolase (sEH) also has been reported to have anti-inflammatory effects in vivo including reduced leukocyte recruitment to the lung. We tested the hypothesis that the in vivo anti-inflammatory effects of sEH inhibitors act through the same mechanisms as the in vitro anti-inflammatory effects of EETs in a rat model of acute inflammation following exposure to tobacco smoke. Contrary to previously published data, we found that sEH inhibition did not reduce tobacco smoke-induced leukocyte recruitment to the lung. Furthermore, sEH inhibition did not reduce tobacco smoke-induced adhesion molecule expression in the lung vasculature. Similarly, concentrations of EETs greater than or equal to their reported effective dose did not reduce TNFα induced expression of the adhesion molecules. These results suggest that the anti-inflammatory effects of sEH inhibitors are independent of leukocyte recruitment and EETs do not reduce the adhesion molecules responsible for leukocyte recruitment in vitro. This demonstrates that the widely held belief that sEH inhibition prevents leukocyte recruitment via EET prevention of adhesion molecule expression is not consistently reproducible.     

The antioxidant and anti-inflammatory activities of tocopherols are independent of Nrf2 in mice

The present study investigated the antioxidant and anti-inflammatory actions of tocopherols in mice and determined whether the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is involved in these activities. A mixture of tocopherols (γ-TmT) that is rich in γ-tocopherol was used. Nrf2 knockout (Nrf2 -/-) and wild-type mice were maintained on 0.03, 0.1, or 0.3% γ-TmT-enriched diet starting 2 weeks before the administration of dextran sulfate sodium (DSS) in drinking water (for 1 week, to induce colonic inflammation), until the termination of the experiment at 3 days after the DSS treatment. Dietary γ-TmT dose dependently lowered the levels of 8-oxo-deoxyguanosine, nitrotyrosine, inflammation index, and leukocyte infiltration in colon tissues, as well as 8-isoprostane and prostaglandin E2 in the serum, in both Nrf2 (-/-) and wild-type mice. No significant difference on the inhibitory actions of γ-TmT between the Nrf2 (-/-) and the wild-type mice was observed. The γ-TmT treatment significantly increased the serum levels of γ- and δ-tocopherols. Interestingly, the serum levels of tocopherol metabolites, specifically the γ- and δ-forms of carboxymethylbutyl hydroxychroman and carboxyethyl hydroxychroman, in Nrf2 (-/-) mice were significantly higher than those in wild-type mice. These findings suggest that the antioxidant and anti-inflammatory activities of γ-TmT in the colon are mostly due to the direct action of tocopherols in trapping reactive oxygen and nitrogen species, independent of the antioxidant enzymes and anti-inflammatory proteins that are regulated by Nrf2; however, Nrf2 knockout appears to affect the serum levels of tocopherol metabolites.     

Alteration of oligomeric state and domain architecture is essential for functional transformation between transferase and hydrolase with the same scaffold

Transferases and hydrolases catalyze different chemical reactions and express different dynamic responses upon ligand binding. To insulate the ligand molecule from the surrounding water, transferases bury it inside the protein by closing the cleft, while hydrolases undergo a small conformational change and leave the ligand molecule exposed to the solvent. Despite these distinct ligand‐binding modes, some transferases and hydrolases are homologous. To clarify how such different catalytic modes are possible with the same scaffold, we examined the solvent accessibility of ligand molecules for 15 SCOP superfamilies, each containing both transferase and hydrolase catalytic domains. In contrast to hydrolases, we found that nine superfamilies of transferases use two major strategies, oligomerization and domain fusion, to insulate the ligand molecules. The subunits and domains that were recruited by the transferases often act as a cover for the ligand molecule. The other strategies adopted by transferases to insulate the ligand molecule are the relocation of catalytic sites, the rearrangement of secondary structure elements, and the insertion of peripheral regions. These findings provide insights into how proteins have evolved and acquired distinct functions with a limited number of scaffolds.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

不同微生物菌剂对基质酶活性和番茄产量及品质的影响

以鸡粪配方基质(腐熟鸡粪:腐熟玉米秸秆:河沙体积为3:4:3)和牛粪配方基质(腐熟牛粪:腐熟玉米秸秆:河沙体积为3:4:3)为试材,研究了基质中分别添加地福来、酵素菌、EM菌、枯草芽孢杆菌和农用微生物菌剂对基质酶活性和番茄产量及品质的影响.结果表明: 两种配方基质添加微生物菌剂40和60 d时根际基质的脲酶、蔗糖酶和碱性磷酸酶活性均显著提高,番茄植株生长量、果实产量及维生素C含量显著高于对照.鸡粪和牛粪配方基质均以添加地福来效果最好,番茄单株产量分别较各自对照增加14.7%和40.0%,果实维生素C含量分别提高22.2%和39.7%.在不添加微生物菌剂的情况下,鸡粪配方基质栽培的番茄单株产量和果实维生素C含量高于牛粪配方基质;分别添加地福来后,两种基质栽培的番茄单株产量和果实维生素C含量无显著差异.     

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences.

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.     

蛹虫草固体发酵大豆基质的成分及抗氧化活性变化研究

以大豆为培养基质,对蛹虫草固体发酵过程中的pH值、淀粉酶、蛋白酶、总糖、还原糖、总酚、ABTS自由基清除率和FRAP进行测定,分析各种物质含量及抗氧化活性变化。结果表明,发酵过程中pH值、淀粉酶和蛋白酶先增加后减少,还原糖含量随着发酵时间延长不断下降,可溶性总糖先减少后增加。总酚含量、ABTS自由基清除率和FRAP值随着发酵时间延长先减少后增加,22d达最大值。发酵成品有望开发成为食品基料及抗氧化食品。     

Catalytic and binding poly-reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution

It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit some activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins-serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities-in the shape of the cavities and the position of catalytic lysine residues-both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active-site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind- inefficient and rudimentary as they are-can provide a considerable selective advantage and a starting point for the evolution of new functions.     

胺鲜酯对高产夏玉米产量及叶片光合羧化酶和保护酶活性的影响

采用田间小区试验,以高产玉米新品种登海661为材料,研究了拔节期叶面喷施10、20和40 mg·L^-1的胺鲜酯（DA-6）对玉米叶片光合羧化酶、保护酶活性和产量的影响.结果表明：喷施胺鲜酯各处理玉米分别比对照（含有表面活性剂和水）增产10.0%(10 mg·L^-1)、8.9%(20 mg·L^-1)和9.4%(40 mg·L^-1),增产效果显著,但各浓度间差异不显著.胺鲜酯处理后,花后玉米的叶面积指数、光合速率、RuBP羧化酶和PEP羧化酶活性均显著上升(P＜0.05),且对光合速率、RuBP羧化酶和PEP羧化酶活性的影响随着处理浓度的增加而提高;与对照相比,胺鲜酯处理后吐丝期、灌浆期、乳熟期和蜡熟期叶片SOD、CAT、POD和GSTs活性及可溶性蛋白质含量显著提高(P＜0.05),MDA含量显著降低(P＜0.05),其中CAT活性随着处理浓度的增加呈上升趋势,其余生理指标各浓度间无显著性差异.     

Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family

Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.     

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes

The C(4) photosynthetic pathway involves the assimilation of CO(2) by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C(4) acids. The enzymes of the CO(2) concentrating mechanism could be affected under water deficit and limit C(4) photosynthesis. Three different C(4) grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C(4) acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C(4) enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L: -malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO(2) by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.     

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5‐aminoimidazole‐4‐carboxamideribotide. Solution NMR spectroscopy of ammonium chloride‐titrated samples was used to study the interaction of NH₃ with amino acids inside this channel. Although numerous residues showed ¹⁵N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78—which is located in the central channel—shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using ¹H‐¹⁵N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon‐induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N′‐[(5′‐phosphoribulosyl)formimino]‐5‐aminoimidazole‐4‐carboxamide‐ribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central β‐barrel of HisF and overlaps with the putative ammonia transport channel.     

The EBG system of E. coli: origin and evolution of a novel beta-galactosidase for the metabolism of lactose

The EBG system of E. coli has served as a model for the evolution of novel functions. This paper reviews the experimental evolution of the catabolism of -galactoside sugars in strains of E. coli that carry deletions of the classical lacZ -galactosidase gene. Evolution of the ebgA encoded Ebg -galactosidase for an expanded substrate range, evolution of the ebgR encoded Ebg repressor for sensitivity to an expanded range of inducers, the amino acid replacements responsible for those changes, and the evolutionary potential of the system are discussed. The EBG system has also served as a model for studying the detailed catalytic consequences of experimental evolution at the physical–chemical level. The analysis of free-energy profiles for the wildtype and all of the various evolved Ebg enzymes has permitted rejection of the Albery–Knowles hypothesis that relates likely changes in free-energy profiles to evolutionary change.     

Evolutionary Morphing of Tryptophan Synthase: Functional Mechanisms for the Enzymatic Channeling of Indole

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions–deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape “minima” in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Carboxymethylation of the enzyme: threonine binding and inhibition are functionally dissociable.

The inactivation of the aspartokinase I-homoserine dehydrogenase I by iodoacetic acid and the effect on the sensitivity to its inhibitor, L-threonine, were examined. Both aspartokinase and homoserine dehydrogenase inactivation, as well as the dehydrogenase desensitization toward L-threonine occur as a pseudo-first order process. During its inactivation, the aspartokinase remains sensitive to L-threonine. At 50% inactivation, the inhibition curve of the aspartokinase by L-threonine displays homotropic cooperative effects. This alkylated protein retains eight binding sites for L-threonine. During the carboxymethylation, the protein remains in the tetrameric form until half of the kinase activity is lost. At the end of the inactivation aggregate forms and dimers appear.     

The mitogenic activities of phosphatidate are acyl-chain-length dependent and calcium independent in C3H/10T1/2 cells.

Phosphatidates (PA or phosphatidic acid) were shown to have mitogenic properties, including the stimulation of DNA synthesis and calcium mobilization in C3H/10T1/2 cells. Their continuous presence for a minimum of 7 h induced DNA synthesis with kinetics similar to that observed when 10% fetal bovine serum was used as a mitogen. PAs with long chain saturated fatty acid moieties were more mitogenic, in a dose-dependent fashion, than PAs with short saturated or unsaturated fatty acid moieties. When compared with lysostearoyl-PA (LSPA), distearoyl-PA (DSPA) was as potent with respect to the induction of DNA synthesis. Lysooleoyl-PA (LOPA) was slightly more potent than dioleoyl-PA (DOPA), but much weaker than DSPA and LSPA. Preincubation with dilauroyl-PA (DLPA) reduces the mitogenic effect of DSPA by 85%. The pattern of mitogenic inhibition suggests that a chain-length-independent, yet PA-specific, mechanism is involved. Both DSPA and DLPA are equally taken up by the cells after 30 min. LOPA, but not LSPA, produced a large calcium transient (1.3 microM), which we found to be derived from intracellular sources. DSPA, the most mitogenic PA tested, produced a weaker transient (0.6 microM). Interestingly, LSPA did not produce any detectable calcium transient. These results suggest that the chain-length-specific step in the signaling mechanism of PA occurs after the initial chain-length-independent partitioning and/or binding to the membrane and that the induction of DNA synthesis is not related to the observed calcium transients.     

杉木和香樟酸雨酸解底物的分解格局

采用凋落物分解袋法,选取湘西地区两种人工林优势树种(香樟和杉木)的凋落叶作为分解材料,分析了两种凋落叶经酸解处理后凋落物分解及其微生物活性的变化。结果表明:酸解处理过程会使两种凋落叶损失一定的质量,随着酸解强度的增加质量损失增加,且酸解处理对香樟凋落物质量损失的影响较杉木凋落物大。不同物种凋落物对酸解强度的差异性反应产生了后续分解过程的差异格局:酸雨酸解作用的增强抑制了杉木凋落物分解过程中包括真菌生物量以及纤维素酶与木质素酶在内的微生物活性;而对于香樟凋落物分解过程,微生物活性对酸雨酸解的响应因变量不同、分解期不同而存在差异性。两物种凋落物的总失重率、木质素和纤维素分解率对酸解作用的响应及其在不同分解期的表现也存在差异性:对于杉木凋落物,在分解前期其失重率表现为T1T2T3,而在后期随酸解强度的增大而升高,即T3T2/T1;香樟凋落物在分解的前期(T1T2T3)与后期(T1T2T3)情况则正好与杉木凋落物相反。总之,酸雨酸解凋落物不仅使底物有机组成发生了变化,在一定程度上导致凋落物物理结构紧密程度改变,而且也可能相应地改变了凋落物定殖微生物群落,这些复合影响从不同程度上决定了凋落物分解及其微生物活性对凋落物底物酸解的响应。