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Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase.
Protein Expr Purif. 2007 Sep; 55(1):189-97.PE

Abstract

Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.

Authors+Show Affiliations

Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226001, India. sarita.sharma05@gmail.comNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

17540580

Citation

Sharma, Sarita, and Vinod Bhakuni. "Cloning and Structural Analysis of Mycobacterium Leprae Serine Hydroxymethyltransferase." Protein Expression and Purification, vol. 55, no. 1, 2007, pp. 189-97.
Sharma S, Bhakuni V. Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase. Protein Expr Purif. 2007;55(1):189-97.
Sharma, S., & Bhakuni, V. (2007). Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase. Protein Expression and Purification, 55(1), 189-97.
Sharma S, Bhakuni V. Cloning and Structural Analysis of Mycobacterium Leprae Serine Hydroxymethyltransferase. Protein Expr Purif. 2007;55(1):189-97. PubMed PMID: 17540580.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase. AU - Sharma,Sarita, AU - Bhakuni,Vinod, Y1 - 2007/04/29/ PY - 2007/03/01/received PY - 2007/04/12/revised PY - 2007/04/21/accepted PY - 2007/6/2/pubmed PY - 2007/11/2/medline PY - 2007/6/2/entrez SP - 189 EP - 97 JF - Protein expression and purification JO - Protein Expr. Purif. VL - 55 IS - 1 N2 - Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/17540580/Cloning_and_structural_analysis_of_Mycobacterium_leprae_serine_hydroxymethyltransferase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(07)00118-0 DB - PRIME DP - Unbound Medicine ER -