Molecular cloning, expression, purification, and characterization of fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis--a novel Class II A tetramer.
The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.
Ramsaywak
Peggy C
PC
Department of Chemistry, University of Waterloo, 200 University Avenue W, Waterloo, Ont., N2L 3G1, Canada.
Labbé
Geneviève
G
Siemann
Stefan
S
Dmitrienko
Gary I
GI
Guillemette
J Guy
JG
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase