RESUMO
Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.
Assuntos
Hexosaminas/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ácido Aspártico/química , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dimerização , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Glicoconjugados/química , Glicoproteínas/química , Humanos , Ácido N-Acetilneuramínico/química , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Zinco/químicaRESUMO
Lactococcus lactis IL1403 is a lactic acid bacterium that is used widely for food fermentation. Copper homeostasis in this organism chiefly involves copper secretion by the CopA copper ATPase. This enzyme is under the control of the CopR transcriptional regulator. CopR not only controls its own expression and that of CopA, but also that of an additional three operons and two monocistronic genes. One of the genes under the control of CopR, yahD, encodes an α/ß-hydrolase. YahD expression was induced by copper and cadmium, but not by other metals or oxidative or nitrosative stress. The three-dimensional structure of YahD was determined by X-ray crystallography to a resolution of 1.88 Å. The protein was found to adopt an α/ß-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. Functional testing of YahD for a wide range of substrates for esterases, lipases, epoxide hydrolases, phospholipases, amidases and proteases was, however, unsuccessful. A copper-inducible serine hydrolase has not been described previously and YahD appears to be a new functional member of this enzyme family.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica , Hidrolases/química , Hidrolases/metabolismo , Lactococcus lactis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cádmio/metabolismo , Catálise , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Hidrolases/genética , Lactococcus lactis/química , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Óperon , Alinhamento de SequênciaRESUMO
A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer.
Assuntos
Metiltransferases/química , Peptídeo Sintases/química , Catálise , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Epitopos , Escherichia coli/enzimologia , Fermentação , Fusarium/enzimologia , Cinética , Espectroscopia de Ressonância Magnética , Marcadores de Fotoafinidade , Proteínas Recombinantes/química , S-Adenosilmetionina , Saccharomyces cerevisiae/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.