Lipoxygenases and atherosclerosis: protection versus pathogenesis

15 lipoxygenase (15LO) is a lipid-oxidizing enzyme that is considered to contribute to the formation of oxidized lipids in atherosclerotic lesions. Monocyte-macrophages are the key cells that express 15LO in atherosclerotic lesions. In this review, we examine the evidence for 15LO involvement in atherogenesis and explore and evaluate the potential mechanisms whereby 15LO may contribute to the oxidation of LDL by monocyte-macrophages. We also describe several possible pro- versus anti-atherogenic functions that may be mediated by various products of 15LO lipid oxidation. Central pathways involved in regulating 15LO expression and activity that may serve as future targets for intervention and regulation of this enzyme are also reviewed.     

Post-translational modifications of ATP synthase in the heart: biology and function

The ATP synthase complex is a critical enzyme in the energetic pathways of cells because it is the enzyme complex that produces the majority of cellular ATP. It has been shown to be involved in several cardiac phenotypes including heart failure and preconditioning, a cellular protective mechanism. Understanding the regulation of this enzyme is important in understanding the mechanisms behind these important phenomena. Recently there have been several post-translational modifications (PTM) reported for various subunits of this enzyme complex, opening up the possibility of differential regulation by these PTMs. Here we discuss the known PTMs in the heart and other mammalian tissues and their implication to function and regulation of the ATP synthase.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

Cyanide formation from glyoxylate and hydroxylamine catalysed by extracts of higher-plant leaves

Extracts of spinach, maize and barley contain an enzyme which catalyses the formation of hydrogen cyanide from glyoxylate and hydroxylamine. The enzyme is dependent upon ADP and a divalent cation such as manganese. Glyoxylicacid oxime is a poor substrate for the enzyme. Carbon dioxide is another product of the reaction and is probably produced in 1:1 stoichiometry with hydrogen cyanide. The possible relationship of this enzyme to the regulation of nitrate reduction is discussed.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation.

1. Melanosomal tyrosinase was isolated from normal C57B1 mice, and a comparison of the tyrosine-hydroxylation and dopa (3,4-dihydroxyphenylalanine)-oxidation activities of this enzyme was made. 2. The results indicate that in the absence of dopa cofactor, this enzyme is capable of tyrosine hydroxylation, but with very little subsequent dopa oxidation and melanin formation. 3. This mechanism of enzyme action may play an important role in the intracellular regulation of melanin formation. 4. Further, dopa appears to act as a positive allosteric effector for tyrosine hydroxylation by tyrosinase, in addition to its known activity as a hydrogen donor for the reaction.     

Brain formation of oestrogen in the mouse: Sex dimorphism in aromatase development

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase, converting testosterone (T) to oestradiol-17β (E₂), is a key enzyme in the brain and regulation of this enzyme is likely to determine availability of E₂ effective for neural differentiation. In rodents, oestrogens are formed very actively during male perinatal brain development. This paper reviews work on the sexual differentiation of the brain aromatase system in vitro. Embryonic day 15 mouse hypothalamic culture aromatase activity (AA: mean V_max = 0.9 pmol/h/mg protein) is several times greater than in the adult, whereas apparent K_m is similar for both (30–40 nM). Using microdissected brain areas and cultured cells of the mouse, sex differences in hypothalamic AA during both early embryonic and later perinatal development can be demonstrated, with higher E₂ formation in the male than in the female. The sex differences are brain region-specific, since no differences between male and female are detectable in cultured cortical cells. AA quantitation and immunoreactive staining with an aromatase polyclonal antibody both identify neuronal rather than astroglial localizations of the enzyme. Kainic acid eliminates the gender difference in hypothalamic oestrogen formation indicating, furthermore, that this sex dimorphism is neuronal. Gender-specific aromatase regulation is regional in the brain. Oestrogen formation is specifically induced in cultured hypothalamic neurones of either sex by T, since androgen has no effect on cortical cells. Androgen is clearly involved in the growth of hypothalamic neurones containing aromatase. It appears that differentiation of the brain involves maturation of a gender-specific network of oestrogen-forming neurones.     

The structure and allosteric regulation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a ‘housekeeping’ enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an ‘antenna’ like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.     

Enzymes of secondary metabolism and the biosynthesis of macrolide antibiotics

Enzymes participating in the biosynthesis of macrolide antibiotics are reviewed. Enzyme activities are known to play a pivotal role in the formation of biologically active compounds. Hence it is essential to understand these enzymes, their properties and regulation. Macrolide antibiotics represent a relatively compact group of natural products and include several excellent model compounds suitable for enzyme studies that could be generalized to other oligoketide antibiotics.     

A kinetic scheme of human neutrophil 5-lipoxygenase activity

In animal cells arachidonic acid is metabolized via the 5-, 12- and 15-lipoxygenase pathways. The kinetic mechanism of action of plant (soya) and animal (reticulocyte) 15-lipoxygenases is now well established. 5-Lipoxygenase possesses, in all probability, the most complex mechanism of activity regulation. At present several effectors of neutrophil 5-lipoxygenase, both cytosolic and membrane-bound ones, have been identified. The molecular and kinetic mechanisms of action of the enzyme are still open to question. A kinetic scheme of regulation of synthesis of arachidonic acid 5-lipoxygenase metabolites which does not exclude the presence of two binding sites on the enzyme molecule, is proposed. Within the framework of this kinetic scheme the enzyme activator complex may be the active form of the enzyme. There is evidence that the curve for the time dependence of 5-HETE accumulation in neutrophils stimulated by the Ca2+ ionophore A23187 has a maximum, while the corresponding curve for the LTB4 accumulation is a curve with saturation. It was shown that an increase in the concentration of exogenous arachidonate induces the synthesis of 5-HETE, whereas the concentration of LTB4 remains practically unchanged. The results of mathematical analysis of the above kinetic scheme and a comparison of experimental and calculated values suggest that the reaction effector, Ca2+, plays a crucial regulatory role in the observed kinetic dependencies reflecting the formation of two sequential products of 5-lipoxygenase oxidation of arachidonate. In this way Ca2+ strongly influences the first step of the reaction, i.e., 5-HETE formation; its effect on the second reaction step (5-HETE conversion into LTA4) is far less apparent.     

Probing the active site of homoserine trans-succinylase

Homoserine trans-succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction. The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis. This complex regulation reflects the key role of this enzyme in bacterial metabolism. Here, we demonstrate--using proteomics and high-resolution mass spectrometry--that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46. Replacing these lysine residues by alanine abolished the enzymatic activity. These findings position the lysine residues, one of which is conserved, at the active site.     

HO in pregnancy

The enzyme heme oxygenase (HO) has been implicated in several physiological functions throughout the body including control of vascular tone and regulation of the inflammatory and apoptotic cascades as well as contributing to the antioxidant capabilities in several organ systems. These various properties attributed to HO are carried out through the catalytic products of heme degradation, namely carbon monoxide (CO), biliverdin, and free iron (Fe2+). As the newly emerging roles of HO in normal organ function have come to light, researchers in several disciplines have assessed the role of this enzyme in various physiological and pathological changes taking place in the human body over a lifetime. Included in this new wave of interest is the involvement of HO, and its by-products, in the normal function of the vital organ of pregnancy, the placenta. In this review the role of HO, and its catalytic products, will be examined in the context of pregnancy. The different isoforms of the HO enzyme (HO-1, HO-2, HO-3) have been localized throughout placental tissue, and have been shown to be physiologically active. The HO protein and more specifically its catalytic by-products (CO, biliverdin, and Fe2+) have been postulated to be involved in the maintenance of uterine quiescence throughout gestation, regulation of hemodynamic control within the uterus and placenta, regulation of the apoptotic and inflammatory cascades in trophoblast cells, and the maintenance of a balance of the oxidant-antioxidant status within the placental tissues. The association between this enzyme system, and its above-noted roles throughout pregnancy, with the hypertensive disorder of pregnancy preeclampsia (PET), will also be examined. It is hypothesized that a decrease in HO expression and/or activity throughout gestation would be capable of initiating several pathological processes involved in the etiology of PET. This hypothesis has led to further discussion emphasizing the possibility of novel therapeutic designs targeting this enzyme system for the treatment of PET.     

Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase

Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.     

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q_p) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q_p(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed‐batch experiments, leads to errors in the prediction, because q_p is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate‐limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin‐N synthase (IPNS) was the main rate‐limiting enzyme for penicillin‐G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady‐state as well as the dynamics during chemostat start‐up and fed‐batch cultivation. Biotechnol. Bioeng. 2010;106: 608–618. © 2010 Wiley Periodicals, Inc.     

When cleavage is not attractive: non-catalytic inhibition of ubiquitin chains at DNA double-strand breaks by OTUB1

Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes.     

胆固醇合成途径的关键酶：HMG辅酶A还原酶和疾病

在真核生物中,３羟基３甲基戊二酸单酰辅酶Ａ还原酶是催化合成胆固醇和非甾醇类异戊二烯的共同前体———甲羟戊酸的关键酶。该酶的活性在转录、转录后、翻译及酶降解等多个水平上受到调节。胆固醇在动脉粥样硬化的发生、发展中起重要作用,而异戊二烯则参与细胞增殖调节、信号转导及肿瘤发生过程。目前,该酶已成为一些有效的抗动脉粥样硬化药物治疗的靶点。