Literature summary for 2.4.1.140 extracted from

Molina, M.; Moulis, C.; Monties, N.; Pizzut-Serin, S.; Guieysse, D.; Morel, S.; Cioci, G.; Remaud-Simeon, M.
Deciphering an undecided enzyme investigations of the structural determinants involved in the linkage specificity of alternansucrase (2019), ACS Catal., 9, 2222-2237 .

No PubMed abstract available

Search for more literature for 2.4.1.140

Application

Application	Comment	Organism
synthesis	alternan polymer is more water soluble and less viscous than dextrans, making it a good substitute for Arabic gum. Nanoparticles and films have been obtained with alternan produced by Leuconostoc citreum ABK-1 alternansucrase, thereby opening up other potential applications in nanotechnology	Leuconostoc citreum

Cloned(Commentary)

Cloned (Comment)	Organism
gene asr, recombinant expression of wild-type enzyme and truncated mutants in Escherichia coli strain BL21(DE3)	Leuconostoc citreum

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant truncated mutant ASRDELTA2 (ASR (Met1-Ala2057)), sitting drop vapour diffusion method, mixing of 200 nl of 8 mg/ml protein in 30 mM MES buffer, pH 6.5, 100 mM NaCl, 0.05 g/l CaCl2, and 10 mM isomaltohexaose, with 200 nl of reservoir solution containing 17% w/v PEG 3350, and NaNO3 0.5 M, 3 months, at 12°C, X-ray diffraction structure determination and analysis at 2.8 A resolution	Leuconostoc citreum

Crystallization (Comment)

Organism

purified recombinant truncated mutant ASRDELTA2 (ASR (Met1-Ala2057)), sitting drop vapour diffusion method, mixing of 200 nl of 8 mg/ml protein in 30 mM MES buffer, pH 6.5, 100 mM NaCl, 0.05 g/l CaCl2, and 10 mM isomaltohexaose, with 200 nl of reservoir solution containing 17% w/v PEG 3350, and NaNO3 0.5 M, 3 months, at 12°C, X-ray diffraction structure determination and analysis at 2.8 A resolution

Leuconostoc citreum

Protein Variants

Protein Variants	Comment	Organism
additional information	generation of a truncated version of alternansucrase, mutant ASRDELTA2 (ASR (Met1-Ala2057)), three-dimensional structure determination and analysis. Unusual repeated sequences named APY repeats are identified at the C-terminal end of the protein. Their deletion in the mutant ASR C-APY-del (Met1-Gly1425) does not impact the product profile but significantly increases the enzyme half-life time at 50°C by 5fold compared to the full-length ASR. Generation of several truncated forms: ASRDELTA2, ASRDELTA3, ASRDELTA4, and ASRDELTA5, which are deleted, respectively, of the signal peptide (Met1-His38), one predicted disordered region (Met1-Asp78), two predicted disordered regions (Met1-Pro152), and the entire domain V (Met1-Ser398 and Gln1305-Gly1425). Domain organization of ASR truncated mutants, catalytic sites and domain properties of wild-type and mutant enzymes, detailed overview	Leuconostoc citreum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kinetic study conducted with ASR C-APY-del shows that both high molar mass and low molar mass alternan populations are formed in the early stage of the reaction, suggesting that ASR follows a semiprocessive mechanism of polymerization	Leuconostoc citreum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	a calcium ion is present at the interface between domain A and B in interaction with Glu589, Asp595 (motif V), Asp1173 (motif I), and Asn639 (motif II) forming the binding site	Leuconostoc citreum

Organism

Organism	UniProt	Comment	Textmining
Leuconostoc citreum	D3JC16	i.e. Leuconostoc mesenteroides NRRL B-1355	-
Leuconostoc citreum NRRL B-1355	D3JC16	i.e. Leuconostoc mesenteroides NRRL B-1355	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type enzyme and truncated mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration, followed by ultrafiltration	Leuconostoc citreum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 sucrose	-	Leuconostoc citreum	?	-	?
2 sucrose	-	Leuconostoc citreum NRRL B-1355	?	-	?
additional information	for the docking calculations, a model of the ASRDELTA2 glucosyl-enzyme intermediate is constructed based on the high resolution structure of the GH13 covalent intermediate (PDB ID 1S46). Maltose is the best known acceptor for ASR	Leuconostoc citreum	?	-	-
additional information	for the docking calculations, a model of the ASRDELTA2 glucosyl-enzyme intermediate is constructed based on the high resolution structure of the GH13 covalent intermediate (PDB ID 1S46). Maltose is the best known acceptor for ASR	Leuconostoc citreum NRRL B-1355	?	-	-
sucrose + maltose	maltose undergoes alpha-1,6 glucosylation alone to give the oligodextran of DP3 (OD3 panose, alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc), which can be further elongated at either the O6 or the O3 position of the nonreducing unit to give the structures OD4 (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc) and OA4 (alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc), respectively. The oligosaccharide OA4 is quick to appear at the beginning of the reaction (1 min) and accumulates at a much higher level than OD4, indicating that panose is preferentially elongated with an alpha-1,3 linkage. A very small peak of OD5 originating from OD4 only appears toward the end of the reaction (after about 30 min) and is not elongated further (no OD6 is found). As soon as the OA4 starts to accumulate, it is efficiently converts to OA5 (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc). The OA5 itself can act as an acceptor for the formation of two OA6s (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-DGlc and alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc)	Leuconostoc citreum	?	-	?
sucrose + maltose	maltose undergoes alpha-1,6 glucosylation alone to give the oligodextran of DP3 (OD3 panose, alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc), which can be further elongated at either the O6 or the O3 position of the nonreducing unit to give the structures OD4 (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc) and OA4 (alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc), respectively. The oligosaccharide OA4 is quick to appear at the beginning of the reaction (1 min) and accumulates at a much higher level than OD4, indicating that panose is preferentially elongated with an alpha-1,3 linkage. A very small peak of OD5 originating from OD4 only appears toward the end of the reaction (after about 30 min) and is not elongated further (no OD6 is found). As soon as the OA4 starts to accumulate, it is efficiently converts to OA5 (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc). The OA5 itself can act as an acceptor for the formation of two OA6s (alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-DGlc and alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->6)-alpha-D-Glcp-(1->4)-D-Glc)	Leuconostoc citreum NRRL B-1355	?	-	?

Subunits

Subunits	Comment	Organism
More	the catalytic domain A of ASRDELTA2 is formed by a (beta/alpha)8 barrel	Leuconostoc citreum

Synonyms

Synonyms	Comment	Organism
ASR	-	Leuconostoc citreum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
45	-	-	Leuconostoc citreum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5.75	-	assay at	Leuconostoc citreum

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the glycoside hydrolase family 70 (GH70)	Leuconostoc citreum
additional information	analysis of structural determinants involved in the linkage specificity of alternansucrase (ASR), overview. ASR displays two different acceptor subsites in the prolongation of its subsites -1 and +1. The first one is defined by Trp675, a residue of subsite +2, and orients acceptor binding exclusively toward alpha-1,6 linkage synthesis. The second binding site comprises Asp772 and Trp543, two residues defining the +2' and +3' subsites, respectively, which are critical for alpha-1,3 linkage formation. It is proposed that the interplay between these two acceptor sites controls alternance. The enzyme is predicted to adopt the same fold as the other glucansucrases and the same alpha-retaining mechanism involving the contribution of Asp635, Glu673, and Asp767. These amino acids play the role of, respectively, the nucleophile, acid/base catalyst, and transition state stabilizer (TSS) implicated in the formation of the beta-D-glucosyl-enzyme intermediate. Kinetic study conducted with ASR C-APY-del shows that both high molar mass and low molar mass alternan populations are formed in the early stage of the reaction, suggesting that ASR follows a semiprocessive mechanism of polymerization. Three-dimensional structure analysis, overview	Leuconostoc citreum
physiological function	alternansucrase (ASR) is an alpha-transglucosylase capable of catalyzing high and low molar mass alternan, an alpha-glucan comprising alternating alpha-1,3 and alpha-1,6 linkages in its linear chain. ASR also catalyzes transglucosylation from sucrose to many different types of sugar acceptors: methyl-alpha-D-glucoside, maltose, maltodextrin, maltitol, isomaltooligosaccharides, cellobiose, melibiose, raffinose, gentiobiose, and lactose, and produces glucosylated products showing interesting prebiotic properties	Leuconostoc citreum