Literature summary for 2.7.8.6 extracted from

Steiner, K.; Novotny, R.; Patel, K.; Vinogradov, E.; Whitfield, C.; Valvano, M.A.; Messner, P.; Schaeffer, C.
Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a (2007), J. Bacteriol., 189, 2590-2598.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli TOP10 and BL21(DE3) Star cells, and in Salmonella enterica serovar Typhimurium	Geobacillus stearothermophilus
from genomic DNA, (i) in pET28a for inducible expression of entire coding sequence or C-terminal part with N-terminal hexa-His-tag and subcellular localisation in Escherichia coli BL21(DE3) Star, (ii) in pBAD-DEST49: for inducible expression with N-terminal horseradish peroxide-thioredoxin-tag and C-terminal hexa-His-tag in Escherichia coli TOP10, (iii) in pBluescript II SK(+): for complementation studies with full-length WsaP, C-terminal part (aa168-417), N-terminal part (aa1-314) or a mutant lacking the transmembrane domains (aa311-471) in Escherichia coli strain CWG466 and Salmonella enterica strain MSS2	Geobacillus stearothermophilus

Cloned (Comment)

Organism

expressed in Escherichia coli TOP10 and BL21(DE3) Star cells, and in Salmonella enterica serovar Typhimurium

Geobacillus stearothermophilus

from genomic DNA, (i) in pET28a for inducible expression of entire coding sequence or C-terminal part with N-terminal hexa-His-tag and subcellular localisation in Escherichia coli BL21(DE3) Star, (ii) in pBAD-DEST49: for inducible expression with N-terminal horseradish peroxide-thioredoxin-tag and C-terminal hexa-His-tag in Escherichia coli TOP10, (iii) in pBluescript II SK(+): for complementation studies with full-length WsaP, C-terminal part (aa168-417), N-terminal part (aa1-314) or a mutant lacking the transmembrane domains (aa311-471) in Escherichia coli strain CWG466 and Salmonella enterica strain MSS2

Geobacillus stearothermophilus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
membrane	-	Geobacillus stearothermophilus	16020	-
membrane	membrane fraction, differential centrifugation, Western blot	Geobacillus stearothermophilus	16020	-
additional information	no inclusion bodies	Geobacillus stearothermophilus	-	-

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
36500	-	theoretical and apparent for hexa-His-tagged C-terminal part (aa168-417), SDS-PAGE	Geobacillus stearothermophilus
41800	-	hexa-His-tagged full-length enzyme, SDS-PAGE/Western blot	Geobacillus stearothermophilus
56800	-	SDS-PAGE	Geobacillus stearothermophilus
56800	-	theoretical for hexa-His-tagged full-length enzyme	Geobacillus stearothermophilus
72870	-	theoretical for full-length with N-terminal horseradish peroxide-thioredoxin-tag and C-terminal hexa-His-tag, apparent weight slightly less in SDS-PAGE	Geobacillus stearothermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Geobacillus stearothermophilus	initiation step in S-layer glycoprotein glycan biosynthesis	?	-	?
additional information	Geobacillus stearothermophilus NRS 2004/3a	initiation step in S-layer glycoprotein glycan biosynthesis	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Geobacillus stearothermophilus	-	-	-
Geobacillus stearothermophilus	Q7BG44	NRS 2004/3a	-
Geobacillus stearothermophilus NRS 2004/3a	-	-	-
Geobacillus stearothermophilus NRS 2004/3a	Q7BG44	NRS 2004/3a	-

Purification (Commentary)

Purification (Comment)	Organism
from Escherichia coli BL21(DE3) Star and TOP10 membrane preparations, nickel affinity chromatography (batch method), in presence of 0.1 mM lauryldimethylamine N-oxide	Geobacillus stearothermophilus
His-Select nickel affinity gel column chromatography	Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	initiation step in S-layer glycoprotein glycan biosynthesis	Geobacillus stearothermophilus	?	-	?
additional information	K30 antigen biosynthesis by full-length enzyme and N-terminal truncated version, membrane fraction	Geobacillus stearothermophilus	?	-	?
additional information	initiation step in S-layer glycoprotein glycan biosynthesis	Geobacillus stearothermophilus NRS 2004/3a	?	-	?
additional information	K30 antigen biosynthesis by full-length enzyme and N-terminal truncated version, membrane fraction	Geobacillus stearothermophilus NRS 2004/3a	?	-	?
UDP-D-galactose + phosphoryl-polyprenol	glycosyltransferase activity within C-terminus of WsaP, in vitro assay: 1 h, 37°C, pH 8.5, 1 mM EDTA, 10 mM MgCl2, membrane fraction	Geobacillus stearothermophilus	galactosyl-diphospho-polyprenol + CMP	analyses by thin-layer chromatography	?
UDP-D-galactose + phosphoryl-polyprenol	glycosyltransferase activity within C-terminus of WsaP, in vitro assay: 1 h, 37°C, pH 8.5, 1 mM EDTA, 10 mM MgCl2, membrane fraction	Geobacillus stearothermophilus NRS 2004/3a	galactosyl-diphospho-polyprenol + CMP	analyses by thin-layer chromatography	?
UDP-galactose + phosphoryl-polyprenol	-	Geobacillus stearothermophilus	UMP + alpha-D-galactosyl-diphosphopolyprenol	-	?
UDP-galactose + phosphoryl-polyprenol	-	Geobacillus stearothermophilus NRS 2004/3a	UMP + alpha-D-galactosyl-diphosphopolyprenol	-	?

Synonyms

Synonyms	Comment	Organism
UDP-Gal: phosphoryl-polyprenol Gal-1-phosphate transferase	-	Geobacillus stearothermophilus
UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase	-	Geobacillus stearothermophilus
WsaP	-	Geobacillus stearothermophilus
WsaP	the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol	Geobacillus stearothermophilus