User:Drew C. MacKellar/Notebook/ARPA-E/2011/06/27

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KAS engineering
1. Starting to prepare for modifying FabB. Jeff found a paper from the Cronan group (Subrahmanyam 1998) that found overexpression of FabF to be toxic in E. coli, when using the T5 promoter. They were using the same LacI-LacO system found in the Duet vectors, and were unable to titrate the IPTG concentration to achieve expression without toxicity. 10uM IPTG caused overexpression and death; 1uM IPTG caused neither, and intermediate concentrations apparently also caused neither.

I started by fitting the structure of FabB (1EK4; dodecanoate bound) onto FabF (2GFY; also with dodecanoate), to try to see whether the same residues are present in the binding pocket. The residues within 6 angstroms are (those within 6 angstroms of the distal-most carbon are highlighted; most of these are on the opposite dimer, and these are indicated with an asterisk):

S105, G106, G107, G108, P110, Q113*, V134*, A137*, M138*, A139*, S140*, S160, S161, A162, C163, A164, T165, G191, E196, M197, A198, E200, F201, F229, I231, H333, L336, G336

I also made an alignment of FabB with FabF, and highlighted the residues within 6 angstroms of the terminal carbon of dodecanoate in FabB with yellow boxes. I also pointed out the residues chosen for point mutation in FabF with red boxes (shrunk and moved above the alignment, where they would cover up the yellow boxes). Obviously, the residues close to the distal end of the binding pocket are located in very similar positions in the two enzymes. These are located on or near helices 5, 6, 8, and 9 in FabF, and helices 6, 8, 10, and 11 in FabB. These residues have sidegroups that are sticking out away from the helix, and into the protein core or dimer interface, where they can create the binding pocket. These residues are mostly hydrophobic.