Literature summary for 4.2.1.115 extracted from

Piacente, F.; De Castro, C.; Jeudy, S.; Molinaro, A.; Salis, A.; Damonte, G.; Bernardi, C.; Abergel, C.; Tonetti, M.G.
Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars (2014), J. Biol. Chem., 289, 24428-24439 .

Search for more literature for 4.2.1.115

Cloned(Commentary)

Cloned (Comment)	Organism
gene mg534, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of GST-tagged enzyme in Escherichia coli strain BL-21 GOLD, or recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain Rosetta(DE3)	Megavirus chiliensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified detagged enzyme expressed from Escherichia coli strain Rosetta(DE3), hanging drop vapour diffusion method, mixing of 0.001 ml of 6.5 mg/ml protein in 10 mM Tris, pH 8.0, with 500 nl of reservoir solution containing 0.1 M malic acid, and 22-25% PEG 3350 w/v, pH 7.0, at 20°C, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement using the structure of Helicobacter pylori enzyme FlaA1 (PDB entry 2GN4) as search model	Megavirus chiliensis

Crystallization (Comment)

Organism

purified detagged enzyme expressed from Escherichia coli strain Rosetta(DE3), hanging drop vapour diffusion method, mixing of 0.001 ml of 6.5 mg/ml protein in 10 mM Tris, pH 8.0, with 500 nl of reservoir solution containing 0.1 M malic acid, and 22-25% PEG 3350 w/v, pH 7.0, at 20°C, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement using the structure of Helicobacter pylori enzyme FlaA1 (PDB entry 2GN4) as search model

Megavirus chiliensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
UDP-N-acetyl-alpha-D-glucosamine	Megavirus chiliensis	-	UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Megavirus chiliensis	G5CSR9	isolated from marine coastal water off of Chile	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant GST-tagged enzyme from Escherichia coli strain BL-21 GOLD by glutathione affinity chromatography, proteolytic removal of the GST-tag, and ultrafiltration, recombinant His6-tagged enzyme from Escherichia coli strain Rosetta(DE3) by nickel affinity chromatography, proteolytic removal of the His-tag, another step of nickel affinity chromatography, and desalting gel filtration, thereby the recombinant protein corresponds to the native enzyme in which the initial methionine is replaced by GPGS sequence resulting from the cleavage site	Megavirus chiliensis

Purification (Comment)

Organism

recombinant GST-tagged enzyme from Escherichia coli strain BL-21 GOLD by glutathione affinity chromatography, proteolytic removal of the GST-tag, and ultrafiltration, recombinant His6-tagged enzyme from Escherichia coli strain Rosetta(DE3) by nickel affinity chromatography, proteolytic removal of the His-tag, another step of nickel affinity chromatography, and desalting gel filtration, thereby the recombinant protein corresponds to the native enzyme in which the initial methionine is replaced by GPGS sequence resulting from the cleavage site

Megavirus chiliensis

Reaction

Reaction	Comment	Organism	Reaction ID
UDP-N-acetyl-alpha-D-glucosamine = UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O	reaction mechanism	Megavirus chiliensis	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	purified Mg534 protein is incubated in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 25°C, in the presence of 0.2 mM UDP-GlcNAc and 0.1 mM NADP+, GC-MS product identification and analysis	Megavirus chiliensis	?	-	?
UDP-N-acetyl-alpha-D-glucosamine	-	Megavirus chiliensis	UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O	-	?

Synonyms

Synonyms	Comment	Organism
Mg534	-	Megavirus chiliensis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Megavirus chiliensis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Megavirus chiliensis

Cofactor

Cofactor	Comment	Organism	Structure
NADPH	superposition of the NADPH binding domain of Mg534 with CapE highlights a large conformational change of the substrate binding domain that reflects the dynamic of the structure during the catalysis. In absence of the substrate, the domain is rotated and closes the substrate binding pocket	Megavirus chiliensis

General Information

General Information	Comment	Organism
evolution	UDP-GlcNAc inverting 4,6-dehydratases can be divided into two subfamilies, based on the conservation of the canonical YXXXK motif at the active site, like FlaA1, or the presence of an altered motif, MXXXK, where the catalytic tyrosine is replaced by a methionine, represented by CapE. Mg534 is a 4,6-dehydratase 5-epimerase, its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. The Mg534 protein exhibits the canonical catalytic triad containing the 140YXXXK144 motif present in other SDR enzymes	Megavirus chiliensis
metabolism	the first reaction that leads to the formation of the UDP-2-acetamido-2,6-dideoxy-beta-L-hexoses is the dehydration of UDP-D-GlcNAc catalyzed by enzyme Mg534. Mg534 is a 4,6-dehydratase 5-epimerase, an inverting dehydratase. Enzyme Mg535, next in the glycan synthesis pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-L-N-acetylrhamnosamine (UDP-L-RhaNAc). Pathway for the biosynthesis of UDP-2-acetamido-2,6-dideoxy-hexoses, overview	Megavirus chiliensis
additional information	two different types of enzymes are identified. In one case, this step is a simple dehydration with formation of UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose, which is typical of the UDP-bacillosamine or UDP-N-acetyl-D-fucosamine and UDP-N-acetyl-D-quinovosamine pathways. In other cases, the enzymes are inverting 4,6-dehydratases, catalyzing also the epimerization of C-5, with the consequent inversion of the D-configuration to L-, and the formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose. This reaction is catalyzed by PseB/FlaA1 in the case of the pseudaminic acid (Pse) pathway, where the transfer of an amino group to the C-4 carbonyl follows this first step. Superposition of the NADPH binding domain of Mg534 with CapE highlights a large conformational change of the substrate binding domain that reflects the dynamic of the structure during the catalysis. In absence of the substrate, the domain is rotated and closes the substrate binding pocket. Upon incubation with Mg535 and a system for NADPH regeneration, the Mg534 product is converted to a compound with a retention time similar to UDP-GlcNAc	Megavirus chiliensis
physiological function	the enzyme is involved in the Pse pathway for biosynthesis of glycans of the heavily glycosylated fibrils surrounding the viral capsids. Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar. The enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. GC-MS analyses of Megavirus glycoconjugates	Megavirus chiliensis