Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.
6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.
Wu
N
N
Departments of Chemistry, Chemical Engineering, and Biochemistry, Stanford University, Stanford California 94305, USA.
Tsuji
S Y
SY
Cane
D E
DE
Khosla
C
C
D000213:Acyl Carrier Protein / Q000138:chemical synthesis / Q000378:metabolism; D020134:Catalytic Domain; D004579:Electron Transport; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D009097:Multienzyme Complexes; D010455:Peptides