title: Unusual structural, functional, and stability properties of serine hydroxymethyltransferase from Mycobacterium tuberculosis.
abstract: From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5'-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations.
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India.
authors.forename: Sarita
authors.initials: S
authors.lastname: Chaturvedi
authors.forename: Vinod
authors.initials: V
authors.lastname: Bhakuni
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005976:Glutaral / Q000494:pharmacology; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase; D019791:Guanidine; D005976:Glutaral