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1.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 564-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205019

RESUMO

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.


Assuntos
Proteínas de Bactérias/química , N-Acilneuraminato Citidililtransferase/química , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Ácido N-Acetilneuramínico do Monofosfato de Citidina/química , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferase/farmacologia , N-Acilneuraminato Citidililtransferase/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ácidos Siálicos/metabolismo
2.
J Biol Chem ; 260(15): 8838-49, 1985 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-4019457

RESUMO

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.


Assuntos
Glicoproteínas/metabolismo , Ácidos Neuramínicos/metabolismo , Oligossacarídeos/metabolismo , Ácidos Siálicos/biossíntese , Acetilação , Animais , Bovinos , Monofosfato de Citidina/metabolismo , Cavalos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Ácido N-Acetilneuramínico , N-Acilneuraminato Citidililtransferase/isolamento & purificação , N-Acilneuraminato Citidililtransferase/farmacologia , Sialiltransferases/isolamento & purificação , Sialiltransferases/farmacologia , Glândula Submandibular/enzimologia , Suínos
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