The enzymes of sialic acid biosynthesis

The sialic acids are a family of nine carbon alpha-keto acids that play a wide variety of biological roles in nature. In mammals, they are found at the distal ends of cell surface glycoconjugates, and thus are major determinants of cellular recognition and adhesion events. In certain strains of pathogenic bacteria, they are found in capsular polysaccharides that mask the organism from the immune system by mimicking the exterior of a mammalian cell. This review outlines recent developments in the understanding of the two main enzymes responsible for the biosynthesis of the sialic acid, N-acetylneuraminic acid. The first, a hydrolyzing UDP-N-acetylglucosamine 2-epimerase, generates N-acetylmannosamine and UDP from UDP-N-acetylglucosamine. The second, sialic acid synthase, generates either N-acetylneuraminic acid (bacteria) or N-acetylneuraminic acid 9-phosphate (mammals) in a condensation reaction with phosphoenolpyruvate. An emphasis is placed on an understanding of the mechanistic and structural features of these enzymes.     

N-glycolylneuraminic acid conjugates: implications of their absence in mammalian biochemistry

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter's normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.     

Structual comparison of dermatopontin amino acid sequences

Dermatopontin is a tyrosine-rich acidic extracellular matrix protein of 22 kD with possible functions in cellmatrix interactions and matrix assembly. Database of GenBank+EMBL+DDBJ sequences from Nucleotide, Gene, and Expressed Sequence Tag (EST) Divisions was searched with a keyword “dermatopontin” or mouse dermatopontin amino acid sequence. In addition to five mammals previously described, five mammalian, two bird, one fish dermatopontin genes were detected in vertebrates. Additionally, a goat EST was also shown as goat dermatopontin missing 5′-end of the coding region. Moreover, a mRNA sequence of rhesus monkey dermatopontin was identified, but the deduced amino acid sequence was terminated abruptly due to a nonsense codon. For three 6-residue repeat regions (D-R-E/Q-W-X-F/Y) that may function as part of a glycosaminoglycan binding site, the first repeat sequence is D-R-Q-W-N-Y in all mammals while Glutamine is substituted for Leucine in birds. The second and the third repeats are conserved in all vertebrates. The N-Y-D sequence, the consensus in many amine oxidases, is conserved in mammals except rodents. Asparagine is substituted for Threonine in birds. The tetrapeptide R-G-A-T sequence possibly recognizing the integrin family is conserved in mammals and birds, but Alanine was substituted for Glutamine in zebrafish resulting in loss of activity. In conclusion, functionally significant amino acid sequences in vertebrate dermatopontins are conserved in mammals, while they are not necessarily identified in birds and fish. The original function of vertebrate dermatopontins may be glycosaminoglycan binding and functions as a ligand for integrin and an amine oxidase may be gained in the process of evolution.     

Repeated functional convergent effects of NaV1.7 on acid insensitivity in hibernating mammals

Hibernating mammals need to be insensitive to acid in order to cope with conditions of high CO₂; however, the molecular basis of acid tolerance remains largely unknown. The African naked mole-rat (Heterocephalus glaber) and hibernating mammals share similar environments and physiological features. In the naked mole-rat, acid insensitivity has been shown to be conferred by the functional motif of the sodium ion channel Na_V1.7. There is now an opportunity to evaluate acid insensitivity in other taxa. In this study, we tested for functional convergence of Na_V1.7 in 71 species of mammals, including 22 species that hibernate. Our analyses revealed a functional convergence of amino acid sequences, which occurred at least six times independently in mammals that hibernate. Evolutionary analyses determined that the convergence results from both parallel and divergent evolution of residues in the functional motif. Our findings not only identify the functional molecules responsible for acid insensitivity in hibernating mammals, but also open new avenues to elucidate the molecular underpinnings of acid insensitivity in mammals.     

Primer on genes encoding enzymes in sialic acid metabolism in mammals

Sialic acid, a nine-carbon sugar acid usually is present in the non-reducing terminal position of free oligosaccharides and glycoconjugates. Sialylated conjugates in mammals perform important roles in cellular recognition, signaling, host–pathogen interaction and neuronal development. Metabolism of sialylated conjugates involves a complex pathway consisting of enzymes distributed among the different compartments in the cell. These enzymes are encoded by 32 genes diversely distributed throughout the mammalian genome. Genetic variants in some of these genes are associated with embryonic lethality and abnormal phenotypes in mice and neuromuscular diseases, carcinomas and immune-mediated diseases in humans. In humans, the CMP-NeuAc-hydroxylase (CMAH) enzyme is inactivated due to a deletion mutation in the encoded enzyme. This lack of Neu5Gc phenotype makes humans unique among mammals. This review focuses on genes encoding enzymes in sialic acid metabolism pathways in mammalian cells with special emphasis on the human, mouse and cow.     

A second fatty acid amide hydrolase with variable distribution among placental mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.     

On the importance of fatty acid composition of membranes for aging

The membrane pacemaker theory of aging is an extension of the oxidative stress theory of aging. It emphasises variation in the fatty acid composition of membranes as an important influence on lipid peroxidation and consequently on the rate of aging and determination of lifespan. The products of lipid peroxidation are reactive molecules and thus potent damagers of other cellular molecules. It is suggested that the feedback effects of these peroxidation products on the oxidative stress experienced by cells is an important part of the aging process. The large variation in the chemical susceptibility of individual fatty acids to peroxidation coupled with the known differences in membrane composition between species can explain the different lifespans of species, especially the difference between mammals and birds as well as the body-size-related variation in lifespan within mammals and birds. Lifespan extension by calorie-restriction can also be explained by changes in membrane fatty acid composition which result in membranes more resistant to peroxidation. It is suggested that lifespan extension by reduced insulin/IGF signalling may also be mediated by changes in membrane fatty acid composition.     

Molecular mechanisms involved in the adaptation to amino acid limitation in mammals

In mammals, metabolic adaptations are required to cope with episodes of protein deprivation and malnutrition. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of the expression of numerous genes. In particular, it has been shown that amino acids by themselves can modify the expression of target genes. This review describes the regulation of amino acids homeostasis and the their role as signal molecules. The recent advances in the understanding of the molecular mechanisms involved in the control of mammalian gene expression in response to amino acid limitation will be described.     

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.     

D-Amino acid metabolism in mammals: biosynthesis, degradation and analytical aspects of the metabolic study

It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of D-amino acids are present in mammals. The most abundant D-amino acids are D-serine and D-aspartate. D-Serine, which is synthesized by serine racemase and is degraded by D-amino-acid oxidase, is present in the brain and modulates neurotransmission. D-Aspartate, which is synthesized by aspartate racemase and degraded by D-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. D-Serine and D-aspartate bind to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these D-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and D-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on D-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and D-amino-acid oxidases.     

Membrane fatty acid composition of tissues is related to body mass of mammals

Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (2263) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of 3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 2263. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.This work was supported by an Australian Commonwealth Postgraduate Research Scholarship from the University of Wollongong to P. Couture and by a grant from the Australian Research Council to A.J. Hulbert. We wish to thank Voytek Mantaj for technical assistance.     

Amino acid metabolism and the energetics of growth

The nonessential amino acids are involved in a large number of functions that are not directly associated with protein synthesis. Recent studies using a combination of transorgan balance and stable isotopic tracers have demonstrated that a substantial portion of the extra‐splanchnic flux of glutamate, glutamine, glycine and cysteine derives from tissue synthesis. A key amino acid in this respect is glutamic acid. Little glutamic acid of dietary origin escapes metabolism in the small intestinal mucosa. Furthermore, because glutamic acid is the only amino acid that can be synthesized by mammals by reductive amination of a ketoacid, it is the ultimate nitrogen donor for the synthesis of other nonessential amino acids. Because the synthesis of glutamic acid and its product glutamine involve the expenditure of adenosine triphosphate (ATP), it seems possible that nonessential amino acid synthesis might have a significant bearing on the energetics of protein synthesis and, hence, of protein deposition. This paper discusses the topic of the energy cost of protein deposition, considers the metabolic physiology of amino acid oxidation and nonessential amino acid synthesis, and attempts to combine the information to speculate on the overall impact of amino acid metabolism on the energy exchanges of animals.     

The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo beta-oxidation but are first subjected to fatty acid alpha-oxidation in which the terminal carboxyl-group is released as CO(2). The mechanism of alpha-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid alpha-oxidation, and the complete alpha-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid alpha-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Iophenoxic acid as a bait marker for wild mammals: efficacy and safety considerations

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Rat acetoacetic acid decarboxylase inhibition by acetone

Although in mammals, acetone formation from acetoacetic acid is normally regarded as a non-enzymatic (spontaneous) process, the existence of an acetoacetic acid decarboxylase activity was postulated recently. The results imply that this enzymatic activity can be relatively important at the physiological concentrations of ketone bodies found in the rat and that acetone acts as a competitive inhibitor of this enzyme.     

Penicillin inhibitors of purple acid phosphatase

Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.     

The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid

Metabolic adaptation is required to cope with episodes of protein deprivation and malnutrition. GCN2 eIF2alpha kinase, a sensor of amino acid deficiency, plays a key role in yeast and mammals in modulating amino acid metabolism as part of adaptation to nutrient deprivation. The role of GCN2 in adaptation to long-term amino acid deprivation in mammals, however, is poorly understood. We found that expression of lipogenic genes and the activity of fatty acid synthase (FAS) in the liver are repressed and lipid stores in adipose tissue are mobilized in wild-type mice upon leucine deprivation. In contrast, GCN2-deficient mice developed liver steatosis and exhibited reduced lipid mobilization. Liver steatosis in Gcn2(-/-) mice was found to be caused by unrepressed expression of lipogenic genes, including Srebp-1c and Fas. Thus, our study identifies a novel function of GCN2 in regulating lipid metabolism during leucine deprivation in addition to regulating amino acid metabolism.     

Evolutionary loss of ascorbic acid synthesizing ability

The ability to synthesize ascorbic acid has been found only in terrestrial vertebrates. The ability is not present in certain passerine birds, in fruit-eating bats, in guinea-pigs and in Anthropoidea. We postulate that these species lost this ability by a neutral evolutionary change that occurred sporadically by mutation. The change was adopted in the genetic make-up of a few groups of birds and mammals that are widely-scattered in phylogeny. Many herbivorous vertebrate species which consume diets high in ascorbic acid have retained the ability to synthesize it, so that its loss does not appear to be adaptive.