Kinetic and Isotopic Characterization of l-Proline Dehydrogenase from Mycobacterium tuberculosis

Abstract

The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate (P5C) in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel-affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 sec−1 and Km values of 5.7 ± 0.8 mM and 3.4 ± 0.3 μM for L-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that hydride transfer from L-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that L-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady state kinetics reveal an overall kred of 88.5 ± 0.7 sec−1, and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.

Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site

“…The C5 atom of THFA, which represents the hydride donor of proline, is poised 3.5 Å from the flavin N5 atom. This arrangement is consistent with a direct hydride transfer mechanism, which has been shown for PutAs (11) and monofunctional PRODHs (12). All of the interacting residues in the GsPutA-THFA and GsPutA-L-lactate complexes are conserved in PutAs and monofunctional PRODHs, and analogous interactions are observed in the THFA complexes of an Escherichia coli PutA PRODH domain construct (13) and a monofunctional PRODH (14).…”

Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by d-arginine dehydrogenase

“…After isomerization of the enzyme-substrate complex, amine deprotonation triggers the oxidation reaction, with cleavages of the NH and CH bonds of D-leucine occurring asynchronously, as suggested by multiple deuterium kinetic isotope effects on the rate constant for flavin reduction [7]. In this respect PaDADH is similar to proline dehydrogenase [8], but different from other flavin-dependent amine oxidases, such as DAAO or monomeric sarcosine oxidase, for which amine oxidation occurs from the anionic substrate [9].…”

High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose‐binding protein as a solubility tag

“…This is particularly interesting because N-terminally His-tagged TtProDH as treated with BOG was found to be a mixture of dimers and monomers [1]. Moreover, several other ProDHs have been described as dimers [21,23,39] while in some cases, the oligomerization state of ProDH was not reported [22,24,26]. SDS-PAGE indicated that the E. coli cells produce two forms of MBP-TtProDH.…”

“…All recombinant expressions of ProDH reported so far include a His-tagged protein and purification by metal affinity chromatography [18,[21][22][23][24][25][26][27]. However, obtaining soluble protein in preparative amounts has remained problematic [21,26]. Furthermore, it has been reported repeatedly that heterologous expression of ProDH in E. coli yields protein aggregates [24,27].…”