Crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate synthase from Rubrobacter xylanophilus.
Rubrobacter xylanophilus is the only Gram-positive bacterium known to synthesize the compatible solute mannosylglycerate (MG), which is commonly found in hyperthermophilic archaea and some thermophilic bacteria. Unlike the salt-dependent pattern of accumulation observed in (hyper)thermophiles, in R. xylanophilus MG accumulates constitutively. The synthesis of MG in R. xylanophilus was tracked from GDP-mannose and 3-phosphoglycerate, but the genome sequence of the organism failed to reveal any of the genes known to be involved in this pathway. The native enzyme was purified and its N-terminal sequence was used to identify the corresponding gene (mpgS) in the genome of R. xylanophilus. The gene encodes a highly divergent mannosyl-3-phosphoglycerate synthase (MpgS) without relevant sequence homology to known mannosylphosphoglycerate synthases. In order to understand the specificity and enzymatic mechanism of this novel enzyme, it was expressed in Escherichia coli, purified and crystallized. The crystals thus obtained belonged to the hexagonal space group P6(5)22 and contained two protein molecules per asymmetric unit. The structure was solved by SIRAS using a mercury derivative.
Sá-Moura
Bebiana
B
IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
Albuquerque
Luciana
L
Empadinhas
Nuno
N
da Costa
Milton S
MS
Pereira
Pedro José Barbosa
PJ
Macedo-Ribeiro
Sandra
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D008364:Mannosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C441639:mannosyl-3-phosphoglycerate synthase