Literature summary for 2.4.2.38 extracted from

Klutts, J.S.; Levery, S.B.; Doering, T.L.
A beta-1,2-xylosyltransferase from Cryptococcus neoformans defines a new family of glycosyltransferases (2007), J. Biol. Chem., 282, 17890-17899.

Cloned(Commentary)

Cloned (Comment)	Organism
from JEC21 cDNA by PCR with primers introducing C-terminal hexa-His tag, in pCR2.1 TOPO for sequencing, in pPGK (cloned between promoter and terminator of phosphoglycerate kinase) for expression in Saccharomyces cerevisiae that lacks endogenous xylosyltransferase activity	Cryptococcus neoformans

Protein Variants

Protein Variants	Comment	Organism
D550A	mutation of first DXD motif to AXD, complete loss of activity, expression in Saccharomyces cerevisiae	Cryptococcus neoformans
D659A	mutation of second DXD motif to AXD, dramatic reduction in activity, expression in Saccharomyces cerevisiae	Cryptococcus neoformans
N141Q	mutation of the N-glycosylation site, molecular weight shift to 79 kDa corresponding to the molecular weight of the unmodified enzyme, no change in activity, expression in Saccharomyces cerevisiae	Cryptococcus neoformans

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	10 mM EDTA does not reduce endogenous activity	Cryptococcus neoformans

Metals/Ions

Metals/Ions	Comment	Organism	Structure
additional information	activity not dependent on divalent cations	Cryptococcus neoformans

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
79000	-	calculated, 694 amino acids	Cryptococcus neoformans
79000	-	hexa-His tagged mutant N141Q, 12% SDS-PAGE	Cryptococcus neoformans
90000	-	hexa-His tagged wild-type and mutants D550A and D659A, 12% SDS-PAGE	Cryptococcus neoformans
90000	-	purified endogenous activity (serotype D), 12% SDS-PAGE and S-300 gel filtration, identity confirmed by mass spectrometry	Cryptococcus neoformans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
additional information	Cryptococcus neoformans	transfer of beta (1,2)-xylose, capsule synthesis	?	-	?
UDP-D-xylose + galactoxylomannan	Cryptococcus neoformans	-	UDP + [beta-D-xylosyl-(1,2)]-galactoxylomannan	-	?
UDP-D-xylose + glucuronoxylomannan	Cryptococcus neoformans	-	UDP + [beta-D-xylosyl-(1,2)]-glucuronoxylomannan	-	?

Organism

Organism	UniProt	Comment	Textmining
Aspergillus clavatus	-	-	-
Aspergillus fischeri	-	-	-
Aspergillus fumigatus	-	-	-
Aspergillus nidulans	-	-	-
Aspergillus terreus	-	-	-
Chaetomium globosum	-	-	-
Coccidioides immitis	-	-	-
Cryptococcus neoformans	-	serotype A (H99)	-
Cryptococcus neoformans	-	serotype D	-
Cryptococcus neoformans	Q5K8R6	serotype D, strain JEC21 (serotype D MATalpha) and strain ags1delta (serotype D MATalpha ags1delta)	-
Fusarium graminearum	-	-	-
Neurospora crassa	-	-	-
Pyricularia grisea	-	-	-
Ustilago maydis	-	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
side-chain modification	N-linked glycosylation site	Cryptococcus neoformans

Purification (Commentary)

Purification (Comment)	Organism
endogenous activity (2967-fold purification) from serotype D ags1delta strain: solubilisation from crude membranes with 1% Triton X-100 (on ice, 30 min), HiPrep 16/10 Q-Sepharose chromatography with NaCl gradient elution (pH 8), activity peaks were pooled and concentrated (spin, 10000 Da cut-off), HiPrep 26/60 Sephacryl S-300 gel filtration chromatography with isocratic gradient elution (pH 8), concanavalin A-Sepharose 4B chromatography with alpha-methyl-mannoside elution (incubation over night, 4°C), HiTrap DEAE chromatorgraphy with NaCl gradient elution (pH 8), alpha-(1,3)-mannobiose agarose resin affinity chromatography with NaCl gradient (pH 6.5), activity peaks were combined and concentrated, overexpressed hexa-His tagged enzyme: solubilisation, Talon Superflow cobalt metal affinity chromatography, elution with 250 mM imidazole, pH 7.5	Cryptococcus neoformans

Purification (Comment)

Organism

endogenous activity (2967-fold purification) from serotype D ags1delta strain: solubilisation from crude membranes with 1% Triton X-100 (on ice, 30 min), HiPrep 16/10 Q-Sepharose chromatography with NaCl gradient elution (pH 8), activity peaks were pooled and concentrated (spin, 10000 Da cut-off), HiPrep 26/60 Sephacryl S-300 gel filtration chromatography with isocratic gradient elution (pH 8), concanavalin A-Sepharose 4B chromatography with alpha-methyl-mannoside elution (incubation over night, 4°C), HiTrap DEAE chromatorgraphy with NaCl gradient elution (pH 8), alpha-(1,3)-mannobiose agarose resin affinity chromatography with NaCl gradient (pH 6.5), activity peaks were combined and concentrated, overexpressed hexa-His tagged enzyme: solubilisation, Talon Superflow cobalt metal affinity chromatography, elution with 250 mM imidazole, pH 7.5

Cryptococcus neoformans

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	transfer of 300 cpm/min/mg by endogenous activity in crude membranes	Cryptococcus neoformans
additional information	-	transfer of 890000 cpm/min/mg by endogenous, purified activity after alpha-(1,3)-mannobiose affinity chromatography	Cryptococcus neoformans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	transfer of beta (1,2)-xylose, capsule synthesis	Cryptococcus neoformans	?	-	?
UDP-D-xylose + D-mannosyl-alpha-(1,3)-D-mannose	membranes or purified enzyme, radiolabelled UDP-xylose, 8.5-12.5 mM alpha-(1,3)-mannobiose (resembles backbone or dimannose moiety of natural substrates), pH 6.5, 4 h, 20°C	Cryptococcus neoformans	UDP + D-xylosyl-beta-(1,2)-D-mannosyl-alpha-(1,3)-D-mannose	product: trisaccharide with xylose moiety linked to reducing mannose of alpha-(1,3)-mannobiose as revealed by mass spectrometry, NMR and methylation linkage analyses, radiolabel detection by scintillation counting or thin layer chromatography and autoradiography or product purification by solid phase extraction	?
UDP-D-xylose + galactoxylomannan	-	Cryptococcus neoformans	UDP + [beta-D-xylosyl-(1,2)]-galactoxylomannan	-	?
UDP-D-xylose + glucuronoxylomannan	-	Cryptococcus neoformans	UDP + [beta-D-xylosyl-(1,2)]-glucuronoxylomannan	-	?

Synonyms

Synonyms	Comment	Organism
CAP3	previous name based on homology to CAP10	Cryptococcus neoformans
cryptococcus xylosyltransferase 1	-	Cryptococcus neoformans
CXT1	-	Cryptococcus neoformans
Cxt1p	-	Cryptococcus neoformans
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Aspergillus nidulans
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Neurospora crassa
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Aspergillus fischeri
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Aspergillus terreus
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Ustilago maydis
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Pyricularia grisea
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Aspergillus clavatus
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Aspergillus fumigatus
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Fusarium graminearum
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Chaetomium globosum
CXT1p homolog	12 to 23% identity to CTX1p from Cryptococcus neoformans serotype D	Coccidioides immitis
CXT1p homolog	group of 5 genes (CAP1, CAP5, CAP2, CAP4, CAP10) with 20 to 30% identity to CTX1p	Cryptococcus neoformans