Biochemistry p. 5009 - 5015 (2013)
Update date:2022-08-11
Topics:
Serrano, Hector
Blanchard, John S.
The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate 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 s-1 and K m 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 the transfer of hydride 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 s-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.
View MoreSuzhou SuKaiLu Chemical Technology Co., Ltd.
Contact:+86-512-62766020
Address:Floor 4, Building 1, Xinyi Pharmaceutical Valley Wisdom Industrial Park, 415 Changyang Street, Suzhou Industrial Park, Jiangsu Province
Dayang Chem (Hangzhou) Co.,Ltd.
website:http://www.dycnchem.com
Contact:+86-571-88938639
Address:9/F, Unit 2 Changdi Torch Building, 259# WenSan Road, Xihu District, Hangzhou City 310012, P.R.China
Xiamen Hisunny Chemical Co.,Ltd
website:http://www.hisunnychem.com
Contact:+86-592-3327115
Address:Unit 603,No.879,Xiahe Road,Meixin Building,Xiamen,China
zhuzhou zhongle chemical co. ltd.
Contact:+86-0731 28228409
Address:Zhuzhou, Hunan, China
zhangjiagang bonded areas banggao co;ltd
website:http://www.shunchangchem.com
Contact:0086-13921972933
Address:Dongsha Chemical Zone.Zhangjiagang, Jiangsu Province
Doi:10.1021/ja1027698
(2010)Doi:10.1016/j.dyepig.2018.11.022
(2019)Doi:10.1021/ol047983f
(2004)Doi:10.1021/jacs.9b01998
(2019)Doi:10.1055/s-2004-836067
(2005)Doi:10.1039/a704812b
(1997)