Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath.
The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co2+ and inhibited by EDTA. The enzyme displays low K(m) to fructose-1,6-bisphosphate (FBP) and higher K(m) to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co2+ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C(1) assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.
Rozova
O N
ON
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Khmelenina
V N
VN
Mustakhimov
I I
II
Reshetnikov
A S
AS
Trotsenko
Y A
YA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase