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J. Biol. Chem., Vol. 281, Issue 51, 39630-39641, December 22, 2006

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Crystal Structures of Salmonella typhimurium Biodegradative Threonine Deaminase and Its Complex with CMP Provide Structural Insights into Ligand-induced Oligomerization and Enzyme Activation^*

Dhirendra K. Simanshu¹, Handanahal S. Savithri², and Mathur R. N. Murthy²³

From the Molecular Biophysics Unit, the Department of Biochemistry, the Indian Institute of Science, Malleswaram, Bangalore, Karnataka 560 012, India

Two different pyridoxal 5'-phosphate-containing L-threonine deaminases (EC 4.3.1.19 [EC] ), biosynthetic and biodegradative, which catalyze the deamination of L-threonine to -ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of L-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to L-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB·CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for L-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.

Received for publication, June 15, 2006 , and in revised form, September 11, 2006.

The atomic coordinates and structure factors (code 2GN0, 2GN1, and 2GN2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the International Collaborative Research Program of the Institute of Protein Research, Osaka University. Data sets for TdcB crystal forms I and II were collected using the in-house x-ray facility for Structural Biology at the Molecular Biophysics Unit, Indian Institute of Science, supported by the Department of Science and Technology (DST) and the Department of Biotechnology (DBT) of the Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Council for Scientific and Industrial Research, Government of India, for a senior research fellowship.

² Supported by the DST and DBT.

³ To whom correspondence should be addressed. Tel.: 91-80-2293-2458; Fax: 91-80-2360-0535; E-mail: mrn{at}mbu.iisc.ernet.in.

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