Kumar Santosh, Sega Steven, Lynn-Barbe Jamie K, Harris Dannika L, Koehn Jordan T, Crans Debbie C, Crick Dean C
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.
Pathogens. 2023 Sep 18;12(9):1171. doi: 10.3390/pathogens12091171.
In , proline dehydrogenase (PruB) and ∆-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 . Highly active enzymes were expressed and purified using a 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-H)] specificity constants 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 value of 120 ± 0.015 µM was determined for P5C, while the 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 channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.
在[具体内容缺失]中，脯氨酸脱氢酶（PruB）和Δ-吡咯啉-5-羧酸（P5C）脱氢酶（PruA）是单功能酶，它们通过中间体P5C和L-谷氨酸-γ-半醛将脯氨酸氧化为谷氨酸。这两种酶对于致病性[具体内容缺失]的复制至关重要。使用[具体表达系统缺失]表达系统表达并纯化了高活性酶。使用天然底物和化学合成类似物对纯化的酶进行了表征。研究了醌电子受体的结构要求。PruB对所有测试的脂醌类似物（萘醌或苯醌）均表现出活性。在利用天然萘醌[MK-9（II-H）]类似物的PruB测定中，甲萘醌类似物的特异性常数比苯醌类似物高一个数量级。此外，首次使用外源化学合成的P5C对分枝杆菌PruA进行了酶学表征。测定P5C的Km值为120±0.015μM，而NAD的Km值为33±4.3μM。此外，脯氨酸竞争性抑制PruA活性和偶联酶测定，这表明[具体内容缺失]通道底物的重组纯化单功能PruB和PruA酶可能会增加代谢通量并保护细菌免受甲基乙二醛毒性的影响。
