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Journal of Biochemistry Advance Access originally published online on May 15, 2008
Journal of Biochemistry 2008 144(3):295-303; doi:10.1093/jb/mvn067
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© 2008 The Japanese Biochemical Society

Characterization of Pyridoxal 5'-Phosphate-Binding Domain and Folding Intermediate of Bacillus subtilis Serine Hydroxymethyltransferase: an Autonomous Folding Domain

Anant Narayan Bhatt1,* and Vinod Bhakuni2

1Division of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Delhi 110 054; and 2Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

*To whom correspondence should be addressed. Tel: 00 91 011 2390 5130, Fax: 00 91 011 2391 9509, E-mail: anant.bhatt{at}indiatimes.com

Received March 8, 2008; Accepted April 17, 2008


  Abstract

The pyridoxal-5'-phosphate-binding domain (PLPbd) of bsSHMT (Bacillus subtilis serine hydroxymethyltransferase) was cloned and over-expressed in Escherichia coli. The recombinant protein was solublized, refolded and purified from inclusion bodies by rapid mixing followed by ion exchange chromatography. Structural and functional studies suggested the native form of the domain, which obtained as a monomer and had similar secondary and tertiary structural properties as when present in the bsSHMT. The domain also binds to the PLP however with slightly lesser affinity than the native enzyme. GdmCl (guanidium chloride)-induced equilibrium unfolding of the recombinant PLP-binding domain showed a single monophasic transition which corresponds with the second phase transition of the GdmCl-induced unfolding of bsSHMT. The results indicate that PLPbd of bsSHMT is an independent domain, which attains its tertiary structure before the dimerization of partially folded monomer and behaves as a single cooperative unfolding unit under equilibrium conditions.

Key Words: autonomous folding domain, conserved domain, PLP-binding domain, refolding and serine hydroxymethyltransferase

Abbreviations: bsSHMT, Bacillus subtilis SHMT; CD, circular dichroism; eSHMT, E. coli SHMT; GdmCl, guanidium chloride; PLP, pyridoxal-5'-phosphate; SEC, size-exclusion chromatography; SHMT, serine hydroxymethyltransferase and for amino acids single-letter abbreviations have used


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