Anil K Joshi, Vangipuram S Rangan, Andrzej Witkowski, Stuart Smith 

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Engineering of an Active Animal Fatty Acid Synthase Dimer with Only One Competent Subunit  Anil K Joshi, Vangipuram S Rangan, Andrzej Witkowski, Stuart Smith  Chemistry & Biology  Volume 10, Issue 2, Pages 169-173 (February 2003) DOI: 10.1016/S1074-5521(03)00023-1

Figure 1 The Classical Textbook Model for the Homodimeric Animal FAS The subunits lie in a fully extended, antiparallel orientation, such that two centers for fatty acid synthesis are formed at the subunit interface (enclosed in dashed boxes) through cooperation of the three N-terminal domains of one subunit with the four C-terminal domains of the opposite subunit. A central core region of approximately 600 residues has no known catalytic function and is thought to play a role in stabilization of the dimer. KS, β-ketoacyl synthase; MAT, malonyl/acetyl transferase; DH, dehydrase; ER, enoyl reductase; KR, β-ketoacyl reductase; ACP, acyl carrier protein; TE, thioesterase. Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)

Figure 2 Location of Knockout Mutations that Compromise Functionality of Each of the Seven Domains of the FAS Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)

Figure 3 Isolation of 7KO/wt FAS Heterodimers Subunits from homodimeric C terminally His6-tagged 7KO and C terminally FLAG-tagged wild-type FAS (0.3 mg/ml each) were randomized to form a mixture of hetero- and homodimeric species and fractionated on anti-FLAG M2 agarose. Unbound His6-7KO-FAS homodimers were eluted with 0.25 M potassium phosphate (pH 7)/10% glycerol. The bound FLAG-wild-type homodimers and His6-7KO /FLAG-wild-type heterodimers were released with FLAG peptide, detected by assaying ketoreductase activity (A), and fractionated on a Ni-NTA column. FLAG peptide was eluted with 0.25 M potassium phosphate (pH 7)/10% glycerol, FLAG-wild-type homodimers were eluted by inclusion of 15 mM imidazole, and His6-7KO/FLAG-wild-type heterodimers were eluted by 100 mM imidazole (B). Imidazole was removed on a 100 kDa cut-off Vivaspin concentrator (Sartorius). Three hours later, a portion of the purified heterodimer preparation was rechromatographed on Ni-NTA to determine whether subunit exchange may have caused reformation of wild-type homodimers (C). Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)

Figure 4 Analysis of FLAG-wt/His6-7KO Heterodimers by Dibromopropanone Crosslinking Tris(2-carboxyethyl)phosphine (1 mM) was added to the heterodimer preparation, imidazole was removed using a Microcon 100 concentrator (Amicon), and the protein was treated with a 2.5-fold molar excess of dibromopropanone [11]. Products were separated by SDS-PAGE using 3% stacking gels and 4% running gels and detected either by Pro-Blue protein staining (A) or Western blotting (B) using either mouse anti-FLAG (lanes 4 and 5) or mouse anti-His6 (lanes 6 and 7) as primary antibodies and alkaline phosphatase-conjugated goat anti-mouse as secondary antibody. Alkaline phosphatase was detected colorimetrically. Lanes 1 and 4, His6-7KO homodimer; lanes 2, 5, and 7, dibromopropanone-treated His6-7KO/FLAG-wild-type heterodimers; lane 3, dibromopropanone-treated FLAG-wild-type homodimer; lane 6, FLAG-wild-type homodimer. Species i corresponds to an unreacted subunit, species ii to an internally crosslinked subunit, species iii to a singly crosslinked dimer, and species iv to a doubly crosslinked dimer. Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)

Figure 5 Comparison of the Thermal Lability of the Homodimeric Wild-Type and 7KO/wt Heterodimeric FASs The two FAS species (100 μg/ml) were incubated in 0.25 M potassium phosphate buffer (pH 7)/1 mM EDTA/1 mM DTT/10% glycerol, and at various time intervals portions of the reaction mixture were removed for assay of β-ketobutyryl-CoA reductase activity. Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)

Figure 6 Cartoons Illustrating the Functional Interactions between Constituent Domains in the FAS (A), 7KO/wild-type heterodimer; (B), wild-type homodimer. The two subunits are distinguished by red and blue coloring, and domains containing knockout mutations are marked with a dark “x”. Domains engaging in functional interactions are identified by arrows. The lower panel in (B) indicates that FAS subunits assume different conformations in the monomeric and dimeric state. Chemistry & Biology 2003 10, 169-173DOI: (10.1016/S1074-5521(03)00023-1)