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A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements.
FEBS J 2007; 274(7):1701-14FJ

Abstract

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.

Authors+Show Affiliations

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17319935

Citation

Carroll, J David, et al. "A Novel Trehalase From Mycobacterium Smegmatis - Purification, Properties, Requirements." The FEBS Journal, vol. 274, no. 7, 2007, pp. 1701-14.
Carroll JD, Pastuszak I, Edavana VK, et al. A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements. FEBS J. 2007;274(7):1701-14.
Carroll, J. D., Pastuszak, I., Edavana, V. K., Pan, Y. T., & Elbein, A. D. (2007). A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements. The FEBS Journal, 274(7), pp. 1701-14.
Carroll JD, et al. A Novel Trehalase From Mycobacterium Smegmatis - Purification, Properties, Requirements. FEBS J. 2007;274(7):1701-14. PubMed PMID: 17319935.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements. AU - Carroll,J David, AU - Pastuszak,Irena, AU - Edavana,Vineetha K, AU - Pan,Yuan T, AU - Elbein,Alan D, Y1 - 2007/02/23/ PY - 2007/2/27/pubmed PY - 2007/8/1/medline PY - 2007/2/27/entrez SP - 1701 EP - 14 JF - The FEBS journal JO - FEBS J. VL - 274 IS - 7 N2 - Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate. SN - 1742-464X UR - https://www.unboundmedicine.com/medline/citation/17319935/A_novel_trehalase_from_Mycobacterium_smegmatis___purification_properties_requirements_ L2 - https://doi.org/10.1111/j.1742-4658.2007.05715.x DB - PRIME DP - Unbound Medicine ER -