From the EFI’s IS Bridging Project, this publication reports how product distributions differ based on the type of detergent used during assay of E-polyprenyl diphosphate synthases. Although this phenomenon had been partially described in a limited number cases previously, the large scale nature of the EFI enabled methodical cataloguing of activity for a number of EFI target enzymes. This work provides an empirical guide for others working with this class of enzymes.
Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and nonionic detergents, we discovered considerable variability. Examination of a series of nonionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain-lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast, incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain-lengths.
Figure 1. Product distributions for GI 19551716, GI 67866738, and GI 53711383 for 2 h incubations in buffer containing TX or CHAPS. (A) Production distributions for GI 19551716 in buffer containing 0.1% TX (lane 1), 2.5 mM CHAPS (lane 2), 5.0 mM CHAPS (lane 3). (B) Production distributions for GI 19551716, GI 67866738, and GI 53711383 in buffer containing 0.1% TX (lane 1), 0.05% TX (lane 2), 0.01% TX (lane 3), 2.0 mM CHAPS (lane 4). aC-terminal His6 tag.
Figure 3. Product distributions for GI 15640461, GI 16131077, GI 19551716, GI 39934115, GI 53711383, and GI 67866738 for 2 h incubations. Part A; lane 1, 0.1% TX; lane 2, 2 mM PEG12E; Part B; lane 1, 0.1% TX; lane 2, 2 mM NEG12E; Part C; lane 1, 0.1% TX; lane 3, 2 mM HEG12E; lane 4, 2 mM OEG12E; Part D; lane 1, 0.1% TX; lane 2; 2 mM OEG14E. aC-terminal His6 tag. bN-terminal His6 tag.
Figure 4. Product distributions 2 h incubations with different ratios of IPP:DMAPP and IPP:FPP. Lane 1, IPP:DMAPP = 4:1, 0.1 % TX; Lane 2, IPP:DMAPP = 1:2, 0.1 % TX; Lane 3, IPP:DMAPP = 1:2, no TX; Lane 4, IPP:FPP = 4:1, 0.1 % TX; Lane 5, IPP:FPP = 1:2, 0.1 % TX; Lane 6, IPP:FPP = 1:2, no TX. Part A; GI 1955176, GI 53711383; Part B; GI 67866738, and GI 77862362. aC-terminal His6 tag. bN-terminal His6 tag.
Figure 5. Product distributions for GI 29831407, GI 39934115, and 67866738 for 2 h incubations in the presence and absence of TX. Part A; IPP/allylic diphosphate = 4:1. Lane 1, FPP, 0.1% TX; Lane 2, FPP, no TX; Lane 3, DMAPP, no TX. Part B; IPP/allylic diphosphate = 4:1. Lane 1, GPP, 0.1% TX; Lane 2, GPP, no TX; Lane 3, DMAPP, no TX. a. C-terminal His6 tag. b. N-terminal His6 tag.
Figure 6. Product distributions for 24 h incubations in the presence and absence of TX where IPP/FPP = 20:1. Part A. GI 19551716, GI 29376566, GI 29831407, GI 39934115, 53711383, and GI 67866738; Part B, GI 15640461, GI 16131077, GI 19551716, GI 52842862, and GI 58578715. Lane 1, no TX; Lane 2, 0.1% TX. aC-terminal His6 tag. bN-terminal His6 tag.
Reprinted with permission from Biochemistry. Copyright 2013 American Chemical Society.