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J. Biol. Chem., Vol. 281, Issue 35, 25076-25088, September 1, 2006

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Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

SPECTROSCOPIC AND KINETIC STUDIES^*

Vikrant M. Bhor, Sagarika Dev¹, Ganga Ramu Vasanthakumar¹, Parimal Kumar¹, Sharmistha Sinha¹, and Avadhesha Surolia²

From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47 [EC] ), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the Mycobacterium tuberculosis KAPA synthase with both L- and D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 M^–1 s^–1) is 5 times slower than that with L-alanine (k = 399.4 M^–1 s^–1). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed D-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10⁴ M^–1 s^–1). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, D-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (K_i = 114.83 µM) as well as 7,8-diaminopelargonic acid synthase (IC₅₀ = 43.9 µM), the next enzyme of the pathway.

Received for publication, May 10, 2006 , and in revised form, June 8, 2006.

^* This work was supported by a grant from the Department of Biotechnology, Government of India (to A. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text and Scheme S1.

¹ These authors contributed equally to the work.

² To whom correspondence should be addressed. Tel.: 91-11-2671-7102; Fax: 91-11-2617-7626; E-mail: surolia{at}nii.res.in.

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