DB code: S00466

RLCP classification	3.103.70020.355 : Transfer
CATH domain	3.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A	Catalytic domain
E.C.	2.7.7.43
CSA
M-CSA
MACiE

CATH domain	Related DB codes (homologues)
3.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A	S00709 S00465 D00417 D00859 D00860 T00415

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P0A0Z8	N-acylneuraminate cytidylyltransferase	EC 2.7.7.43 CMP-N-acetylneuraminic acid synthetase CMP-NeuNAc synthetase CMP-sialic acid synthetase	PF02348 (CTP_transf_3) [Graphical View]

KEGG enzyme name
N-acylneuraminate cytidylyltransferase CMP-sialate pyrophosphorylase CMP-sialate synthase cytidine 5'-monophosphosialic acid synthetase CMP-Neu5Ac synthetase CMP-NeuAc synthetase acylneuraminate cytidyltransferase CMP-N-acetylneuraminate synthetase CMP-N-acetylneuraminate synthase CMP-N-acetylneuraminic acid synthase CMP-NANA synthetase CMP-sialate synthetase CMP-sialic synthetase cytidine 5'-monophospho-N-acetylneuraminic acid synthetase cytidine 5-monophosphate N-acetylneuraminic acid synthetase cytidine monophosphosialic acid synthetase cytidine monophosphoacetylneuraminic synthetase cytidine monophosphosialate pyrophosphorylase cytidine monophosphosialate synthetase acetylneuraminate cytidylyltransferase

KEGG enzyme name

N-acylneuraminate cytidylyltransferase
CMP-sialate pyrophosphorylase
CMP-sialate synthase
cytidine 5'-monophosphosialic acid synthetase
CMP-Neu5Ac synthetase
CMP-NeuAc synthetase
acylneuraminate cytidyltransferase
CMP-N-acetylneuraminate synthetase
CMP-N-acetylneuraminate synthase
CMP-N-acetylneuraminic acid synthase
CMP-NANA synthetase
CMP-sialate synthetase
CMP-sialic synthetase
cytidine 5'-monophospho-N-acetylneuraminic acid synthetase
cytidine 5-monophosphate N-acetylneuraminic acid synthetase
cytidine monophosphosialic acid synthetase
cytidine monophosphoacetylneuraminic synthetase
cytidine monophosphosialate pyrophosphorylase
cytidine monophosphosialate synthetase
acetylneuraminate cytidylyltransferase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P0A0Z8	NEUA_NEIME	CTP + N-acylneuraminate = diphosphate + CMP-N- acylneuraminate.		Cytoplasm.

KEGG Pathways
Map code	Pathways	E.C.
MAP00530	Aminosugars metabolism

Compound table
	Cofactors	Substrates		Products		Intermediates
KEGG-id	C00305	C00063	C00591	C00013	C01064
E.C.
Compound	Magnesium	CTP	N-Acylneuraminate	Pyrophosphate	CMP-N-acylneuraminate
Type	divalent metal (Ca2+, Mg2+)	amine group,nucleotide	amide group,carbohydrate,carboxyl group	phosphate group/phosphate ion	amide group,amine group,carbohydrate,carboxyl group,nucleotide
ChEBI	18420 18420	17677 17677		29888 29888
PubChem	888 888	6176 6176		1023 21961011 1023 21961011

1eyrA	Unbound	Analogue:CDP	Unbound	Unbound	Unbound
1eyrB	Unbound	Analogue:CDP	Unbound	Unbound	Unbound
1eziA	Unbound	Unbound	Unbound	Unbound	Unbound
1eziB	Unbound	Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [9], [10]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1eyrA	ARG 12;LYS 21;ASP 211	ASP 209;ASP 211(magnesium binding)
1eyrB	ARG 12;LYS 21;ASP 211	ASP 209;ASP 211(magnesium binding)
1eziA	ARG 12;LYS 21;ASP 211	ASP 209;ASP 211(magnesium binding)
1eziB	ARG 12;LYS 21;ASP 211	ASP 209;ASP 211(magnesium binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[5]	Fig.5, p.779	1
[9]	p.8195-8196
[10]	Fig.9, p.152-153	1

References
[1]
Resource
Comments
Medline ID
PubMed ID	4620387
Journal	Biochem Soc Symp
Year	1974
Volume	(40)
Pages	87-116
Authors	Schauer R, Buscher HP, Casals-Stenzel J
Title	Sialic acids: their analysis and enzymic modification in relation to the synthesis of submandibular-gland glycoproteins.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID	2544267
Journal	Carbohydr Res
Year	1989
Volume	186
Pages	326-34
Authors	Petrie CR 3rd, Sharma M, Simmons OD, Korytnyk W
Title	Synthesis of analogs of N-acetylneuraminic acid and their effect on CMP-sialate synthase.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID	2559792
Journal	Carbohydr Res
Year	1989
Volume	194
Pages	49-61
Authors	Christian R, Schreiner E, Zbiral E, Schulz G
Title	The side-chain conformations of N-acetyl-7-,8-,9-deoxy-, and -4,7-dideoxy-neuraminic acid and their effect on the activation of CTP:N-acylneuraminic acid cytidylyltransferase.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	2559791
Journal	Carbohydr Res
Year	1989
Volume	194
Pages	c15-8
Authors	Zbiral E, Schreiner E, Christian R
Title	Synthesis of the 4-acetamido-4-deoxy analogue of N-acetylneuraminic acid and its behaviour towards CMP-sialate synthase.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	1731934
Journal	Biochemistry
Year	1992
Volume	31
Pages	775-80
Authors	Ambrose MG, Freese SJ, Reinhold MS, Warner TG, Vann WF
Title	13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	7858974
Journal	Bioorg Med Chem
Year	1994
Volume	2
Pages	669-74
Authors	Lubineau A, Auge C, Gautheron-Le Narvor C, Ginet JC
Title	Combined chemical and enzymatic synthesis of the sialylated non reducing terminal sequence of GM1b glycolylated ganglioside, a potential human tumor marker.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	9702777
Journal	Nat Biotechnol
Year	1998
Volume	16
Pages	769-72
Authors	Gilbert M, Bayer R, Cunningham AM, DeFrees S, Gao Y, Watson DC, Young NM, Wakarchuk WW
Title	The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	9702765
Journal	Nat Biotechnol
Year	1998
Volume	16
Pages	720-1
Authors	Warner TG
Title	Sweet success with tethered enzyme catalysis.
Related PDB
Related UniProtKB
[9]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	11113120
Journal	J Biol Chem
Year	2001
Volume	276
Pages	8190-6
Authors	Mosimann SC, Gilbert M, Dombroswki D, To R, Wakarchuk W, Strynadka NC
Title	Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP.
Related PDB	1ezi 1eyr
Related UniProtKB
[10]
Resource
Comments	Crystal structures of the homologous enzyme
Medline ID
PubMed ID	11545592
Journal	J Mol Biol
Year	2001
Volume	312
Pages	143-55
Authors	Jelakovic S, Schulz GE
Title	The structure of CMP:2-keto-3-deoxy-manno-octonic acid synthetase and of its complexes with substrates and substrate analogs.
Related PDB	1h7e 1h7f 1h7g 1h7h 1h7t
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID	12355462
Journal	Biotechnol Bioeng
Year	2002
Volume	80
Pages	516-24
Authors	Lee SG, Lee JO, Yi JK, Kim BG
Title	Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID	11893746
Journal	J Biol Chem
Year	2002
Volume	277
Pages	19688-96
Authors	Munster AK, Weinhold B, Gotza B, Muhlenhoff M, Frosch M, Gerardy-Schahn R
Title	Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity.
Related PDB
Related UniProtKB

Comments
According to the literature [9], O2 atom of the substrate, NeuAc, acts as a nucleophile, which makes an SN2 attack on the alpha-phosphate of the other substrate, CTP. During the catalysis, the leaving group, pyrophsophate, can be stabilized by Arg12, Lys21, and Mg2+, which seems to bridging the phosphoryl groups of CTP and the conserved residues, Asp209 and Asp211. The literature [9] suggested that, although there is no candidate for the general base, which can activate the nucleophile, NeuAc O2 atom, an ordered water molecule might serve as the general base or a conformational rearrangement might bring a general base within hydrogen-bonding distance of the O2 atom upon the substrate. Meanwhile, another paper [10] on its homologous enzyme, CMP-Kdo synthease, suggested that a water molecule tightly bound to two aspartic acid residues, Asp98 and Asp225, would acts as the general base, which can activate the acceptor hydroxyl group. In fact, Asp211 of this enzyme, corresponds to Asp225 of the homologous enzyme, in terms of the relative position against the nucleotide substrate. Thus, Asp225 may play a role as a general base, which can activate the acceptor O2 atom, through a water molecule. Moreover, the paper [9] also suggested that an ordered water molecule hydrogen-bonding to mainchain oxygen atom of Asn14 can donate a proton to the leaving group, pyrophosphate. Taken together, the reaction proceeds as follows: (1) Asp211 acts as a general base to activate O2 hydroxyl group, through water molecule. (2) The activated O2 atom makes a nucleophilic attack on the alpha-phosphate of CTP. The reaction proceeds via SN2-like mechanism. (3) Arg12 and Lys21 stabilize the leaving pyrophosphate, along with magnesium ion.

Comments

According to the literature [9], O2 atom of the substrate, NeuAc, acts as a nucleophile, which makes an SN2 attack on the alpha-phosphate of the other substrate, CTP. During the catalysis, the leaving group, pyrophsophate, can be stabilized by Arg12, Lys21, and Mg2+, which seems to bridging the phosphoryl groups of CTP and the conserved residues, Asp209 and Asp211.
The literature [9] suggested that, although there is no candidate for the general base, which can activate the nucleophile, NeuAc O2 atom, an ordered water molecule might serve as the general base or a conformational rearrangement might bring a general base within hydrogen-bonding distance of the O2 atom upon the substrate. Meanwhile, another paper [10] on its homologous enzyme, CMP-Kdo synthease, suggested that a water molecule tightly bound to two aspartic acid residues, Asp98 and Asp225, would acts as the general base, which can activate the acceptor hydroxyl group. In fact, Asp211 of this enzyme, corresponds to Asp225 of the homologous enzyme, in terms of the relative position against the nucleotide substrate. Thus, Asp225 may play a role as a general base, which can activate the acceptor O2 atom, through a water molecule.
Moreover, the paper [9] also suggested that an ordered water molecule hydrogen-bonding to mainchain oxygen atom of Asn14 can donate a proton to the leaving group, pyrophosphate.
Taken together, the reaction proceeds as follows:
(1) Asp211 acts as a general base to activate O2 hydroxyl group, through water molecule.
(2) The activated O2 atom makes a nucleophilic attack on the alpha-phosphate of CTP. The reaction proceeds via SN2-like mechanism.
(3) Arg12 and Lys21 stabilize the leaving pyrophosphate, along with magnesium ion.

Created	Updated
2003-07-17	2009-02-26