Mycobacterium tuberculosis Ketopantoate Hydroxymethyltransferase:  Tetrahydrofolate-Independent Hydroxymethyltransferase and Enolization Reactions with α-Keto Acids

Abstract

The panB gene that encodes ketopantoate hydroxymethyltransferase has been cloned from Mycobacterium tuberculosis, expressed, and purified to homogeneity. 1H NMR spectroscopy was used to determine the rate of (i) tetrahydrofolate-independent hydroxymethyltransferase chemistry between formaldehyde and alpha-ketoisovalerate and (ii) deuterium exchange in the methylenetetrahydrofolate-independent enolization of alpha-ketoisovalerate and other alpha-keto acids, catalyzed by PanB. These studies have demonstrated that substrate enolization by PanB is divalent metal-dependent with a preference of Mg2+ > Zn2+ > Co2+ > Ni2+ > Ca2+. The rate of enolization is pH-dependent with optimal activity in the range of 7.0-7.5. The pH profile was bell-shaped, depending on the ionization state of two ionizable groups with apparent pK values of 6.2 and 8.3. Enolization and isotope exchange occurs with some alpha-keto acids (e.g., pyruvate and alpha-ketobutyrate), resulting in the complete exchange of all beta-hydrogens. Enzyme-catalyzed enolization and isotope exchange occur with other long-chain and branched alpha-keto acids, resulting in the stereospecific exchange of only one of the beta-hydrogen atoms. These results are discussed in the context of steric restrictions present in the enzyme active site and the stereochemistry of base-catalyzed isotope exchange.

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

“…In order to gain further support that regulation is mainly carried out at the KPR reaction, we examined the enzymatic properties of KPHMT, which catalyses the first step of the CoA biosynthesis pathway. TK0363 is the only gene that encodes a protein with similarity to the KPHMT proteins from E. coli (Powers and Snell, ; Teller et al ., ; Jones et al ., ), Mycobacterium tuberculosis (Sugantino et al ., ) and A. nidulans (Kurtov et al ., ), with identities of 28%, 23% and 19% respectively. The crystal structures of the KPHMT from E. coli and M. tuberculosis have been elucidated (Chaudhuri et al ., ; Schmitzberger et al ., ; von Delft et al ., ), and residues proposed to be involved in 2‐oxoisovalerate‐binding (Ser46, Lys112, His136 and Glu181) and Mg 2+ ‐binding (Asp45, Asp84 and Glu114) in the KPHMT from E. coli (von Delft et al ., ) are conserved in the TK0363 protein.…”

“…This is similar to the KPHMT from M. tuberculosis . This enzyme can catalyse the KPHMT reaction in the absence of N 5 , N 10 ‐methylenetetrahydrofolate, with a turnover number of 135 min −1 observed with 50 mM 2‐oxoisovalerate and 50 mM free formaldehyde, higher than the k cat value calculated with N 5 , N 10 ‐methylenetetrahydrofolate (47 min −1 ) (Sugantino et al ., ).…”

Coenzyme A biosynthesis: an antimicrobial drug target

“…The crystal structures of the four enzymes have also been determined (Albert et al, 1998;Matak-Vinkovic et al, 2001;von Delft et al, 2001von Delft et al, , 2003Lobley et al, 2005;Ciulli et al, 2007). The Mycobacterium tuberculosis KPHMT, PS and ADC enzymes have been characterized functionally and the crystal structures of KPHMT and PS, and more recently, ADC have been solved (Chopra et al, 2002;Chaudhuri et al, 2003;Sugantino et al, 2003;Wang & Eisenberg, 2003Gopalan et al, 2006). The crystal structure of an ADC from Helicobacter pylori has also been determined (Kwon et al, 2002;Lee & Suh, 2004), and a PS from Staphylococcus aureus was crystallized recently (Seetharamappa et al, 2007).…”

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily,

“…These rate constants are significantly smaller than the k cat values measured for the catalyzed reactions shown in Figure 1 (105 s −1 (5) and 34 s −1 (33), respectively) and thus, the turnover rates greatly exceed the enolization rates. In contrast, the reported k cat for the Mycobacterium tuberculosis MOBH is 47 min −1 and the rate constant for the enolization of α-ketoisovalerate (k enol ) is 752 min −1 (32), which greatly exceeds the turnover rate. The difference in kinetic behavior is consistent with the fact that MOBH catalyzes the enolization of α-ketoisovalerate as the first partial reaction of the overall reaction ( Figure 1) and that MOBH possesses a general base (Glu181) to assist in the catalysis of the α-ketoisovalerate enolization.…”