Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa.
The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.
Muniz
João Renato C
JR
Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Alves
Claudia A
CA
de Pieri
Celina
C
Beltramini
Leila M
LM
Selistre-de-Araújo
Heloisa S
HS
Vettore
André L
AL
da Silva
Felipe R
FR
Arruda
Paulo
P
Garratt
Richard C
RC
Oliva
Glaucius
G
Souza
Dulce H F
DH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D050356:Lipid Metabolism; D008364:Mannosyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D044167:Xylella / Q000201:enzymology*
D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; C093299:GDP-mannosyltransferase; D008364:Mannosyltransferases