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M306192200v1
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Papers In Press, published online ahead of print August 11, 2003
J. Biol. Chem, 10.1074/jbc.M306192200
Submitted on June 12, 2003
Revised on July 28, 2003
Accepted on August 11, 2003

Unusual structural, functional and stability properties of serine hydroxymethyltransferase from mycobacterium tuberculosis

Sarita Chaturvedi and Vinod Bhakuni

Department of Molecular and Structural Biology, CDRI, Lucknow, UP 226001

Corresponding Author: bhakuniv{at}rediffmail.com

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, over-expressed and purified to homogeneity their respective protein products, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homo-dimers of molecular mass about 90kDa 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 one mole of PLP per mole of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains two mole of PLP per mole 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 apo-enzyme but L-cysteine removed the prothestic group, PLP, from both the recombinant enzymes leaving the respective inactive apo-enzymes. 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 guanidium chloride- induced two step unfolding of SHM1 and SHM2 with first step being dissociation of dimer into apo-monomer at low denaturant concentration followed by unfolding of the stabilized monomer at higher denaturant concentration.


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