In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.