. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity. Pantothenate (vitamin B 5 ) is the precursor of the 4Ј-phosphopantetheine moiety of coenzyme A and acyl carrier proteins, which play an important role in metabolism and fatty acid biosynthesis (1-3). The biosynthetic pathway for pantothenate has been elucidated in Escherichia coli and other bacteria and is composed of four enzymes. The first two convert ␣-ketoisovalerate to pantoate, then in a separate branch L-aspartate is decarboxylated to produce -alanine. Finally, pantoate and -alanine are condensed together to form pantothenate (4, 5). The pathway is similar in plants and fungi, although they appear to use a different route to -alanine (6). Bioinformatics analysis has identified the pantothenate pathway as a potential antimicrobial target (7). This is supported by recent genetic studies which show that a pantothenate auxotroph of Mycobacterium tuberculosis fails to establish chronic infections in mice (8).Ketopantoate reductase (KPR, 2 EC 1.1.1.169), encoded by the panE gene, is the second enzyme in the pathway and catalyzes the NADPH-dependent reduction of ketopantoate to pantoate. Previous biochemical studies on the Escherichia coli enzyme established that hydride transfer is stereospecific from the pro-S proton of NADPH to the si face of ketopantoate (Scheme 1A), and the reaction equilibrium favors NADP ϩ and pantoate formation (9, 10). Steady-state kinetic and inhibition analysis are consistent with a sequential ordered bi:bi kinetic mechanism (Scheme 1B) in which NADPH binding is followed by ketopantoate binding, and then pantoate release precedes NADP ϩ release (10). The pH dependence of catalysis is consistent with the involvement of a general acid/base in the catalytic mechanism (9, 10). Site-directed mutagenesis implicated Lys 176 and Glu 256 as important for catalysis (9). The crystal structure of the apoenzyme was solved by MatakVinkovic et al. (11) at 1.7 Å of resolution. KPR belongs to the 6-phosphogluconate dehydrogenase superfamily in the SCOP data base (11,12). Among other enzymes in this superfamily are acetohydroxy acid isomeroreductase (13), short chain L-3-hydroxyacyl-CoA dehydrogenase (14), ⌬ 1 -pyrroline-5-carboxylate reductase (15), and prephenate dehydrogenase (16). The secondary structure of KPR comprises 13 ␣-helices and 11 -strands. The enzyme is monomeric with a molecular mass of 34 kDa and is composed of a coenzyme binding domain and a substrate binding domain separated by a large cleft. The N-terminal domain has an ␣ Rossmann-type fold featured in many nucleotide-binding proteins, with a glycine-rich region ( 7 GCGALG 12 ) for coenzyme recognition (17). The C-terminal substrate binding domain is composed of 8 ␣-helices and has a core of two long antiparallel helices, which is a common motif within the superfamily.
* This work was supported by the Biotechnology an...