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J. Biol. Chem., Vol. 282, Issue 40, 29521-29530, October 5, 2007

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Enzymatic Characterization of Dihydrolipoamide Dehydrogenase from Streptococcus pneumoniae Harboring Its Own Substrate^*

Anders P. Håkansson^¶1 and Alexander W. Smith^||

From the Department of Microbiology and Immunology and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, New York 14214, the ^¶New York State Center of Excellence in Bioinformatics & Life Sciences, Buffalo, New York 14203, and the ^||Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

This study describes the enzymatic characterization of dihydrolipoamide dehydrogenase (DLDH) from Streptococcus pneumoniae and is the first characterization of a DLDH that carries its own substrate (a lipoic acid covalently attached to a lipoyl protein domain) within its own sequence. Full-length recombinant DLDH (rDLDH) was expressed and compared with enzyme expressed in the absence of lipoic acid (rDLDH^-LA) or with enzyme lacking the first 112 amino acids constituting the lipoyl protein domain (rDLDH^-LIPOYL). All three proteins contained 1 mol of FAD/mol of protein, had a higher activity for the conversion of NAD⁺ to NADH than for the reaction in the reverse direction, and were unable to use NADP⁺ and NADPH as substrates. The enzymes had similar substrate specificities, with the K_m for NAD⁺ being 20 times higher than that for dihydrolipoamide. The kinetic pattern suggested a Ping Pong Bi Bi mechanism, which was verified by product inhibition studies. The protein expressed without lipoic acid was indistinguishable from the wild-type protein in all analyses. On the other hand, the protein without a lipoyl protein domain had a 2–3-fold higher turnover number, a lower K_I for NADH, and a higher K_I for lipoamide compared with the other two enzymes. The results suggest that the lipoyl protein domain (but not lipoic acid alone) plays a regulatory role in the enzymatic characteristics of pneumococcal DLDH.

Received for publication, April 13, 2007 , and in revised form, August 1, 2007.

^* This study was supported by the Swedish Cancer Society (to A. P. H.), by National Institutes of Health Grants AI21548 and HL54818, and by National Institutes of Health Immunology Training Fellowship AI07051 (to A. W. S.). The majority of this work was conducted in the Department of Microbiology, University of Alabama at Birmingham, AL, during the post-doctoral studies of A. P. H. in the laboratory of Dr. David E. Briles. 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 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University at Buffalo, SUNY, 145 Biomedical Research Bldg., 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-6058; Fax: 716-829-2158; E-mail: andersh{at}buffalo.edu.

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