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Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis.
Arch Biochem Biophys 2004; 426(2):250-7AB

Abstract

Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, The University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15158675

Citation

Edavana, Vineetha Koroth, et al. "Cloning and Expression of the Trehalose-phosphate Phosphatase of Mycobacterium Tuberculosis: Comparison to the Enzyme From Mycobacterium Smegmatis." Archives of Biochemistry and Biophysics, vol. 426, no. 2, 2004, pp. 250-7.
Edavana VK, Pastuszak I, Carroll JD, et al. Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis. Arch Biochem Biophys. 2004;426(2):250-7.
Edavana, V. K., Pastuszak, I., Carroll, J. D., Thampi, P., Abraham, E. C., & Elbein, A. D. (2004). Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis. Archives of Biochemistry and Biophysics, 426(2), pp. 250-7.
Edavana VK, et al. Cloning and Expression of the Trehalose-phosphate Phosphatase of Mycobacterium Tuberculosis: Comparison to the Enzyme From Mycobacterium Smegmatis. Arch Biochem Biophys. 2004 Jun 15;426(2):250-7. PubMed PMID: 15158675.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis. AU - Edavana,Vineetha Koroth, AU - Pastuszak,Irena, AU - Carroll,J D, AU - Thampi,Prajitha, AU - Abraham,Edathera C, AU - Elbein,Alan D, PY - 2003/12/01/received PY - 2004/02/12/revised PY - 2004/5/26/pubmed PY - 2004/7/20/medline PY - 2004/5/26/entrez SP - 250 EP - 7 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 426 IS - 2 N2 - Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/15158675/Cloning_and_expression_of_the_trehalose_phosphate_phosphatase_of_Mycobacterium_tuberculosis:_comparison_to_the_enzyme_from_Mycobacterium_smegmatis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003986104000931 DB - PRIME DP - Unbound Medicine ER -