Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis.
Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K(m) of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K(i) was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 degrees C and pH 9.0, and did not require mono- or divalent cations for its activity.
Mathur
Divya
D
Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Ahsan
Zaid
Z
Tiwari
Madhulika
M
Garg
Lalit C
LC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D002412:Cations; D005636:Fructosephosphates; D005942:Gluconates; D011994:Recombinant Proteins; D012965:Sodium Chloride; D011189:Potassium Chloride; C027618:fructose-6-phosphate; D004247:DNA; D005956:Glucose-6-Phosphate Isomerase; C008884:6-phosphogluconic acid