Modular polyketide synthases catalyze the biosynthesis of medicinally important natural products through an assembly-line mechanism. Although these megasynthases display very precise overall selectivity, we show that their constituent modules are remarkably tolerant toward diverse incoming acyl chains. By appropriate engineering of linkers, which exist within and between polypeptides, it is possible to exploit this tolerance to facilitate the transfer of biosynthetic intermediates between unnaturally linked modules. This protein engineering strategy also provides insights into the evolution of modular polyketide synthases.
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.
Polyketide synthases (PKSs) have represented fertile targets for rational manipulation via protein engineering ever since their modular architecture was first recognized. However, the mechanistic principles by which biosynthetic intermediates are sequentially channeled between modules remain poorly understood. Here we demonstrate the importance of complementarity in a remarkably simple, repetitive structural motif within these megasynthases that has been implicated to affect intermodular chain transfer [Gokhale, R. S., et al. (1999) Science 284, 482]. The C- and N-terminal ends of adjacent PKS polypeptides are capped by short peptides of 20-40 residues. Mismatched sequences abolish intermodular chain transfer without affecting the activity of individual modules, whereas matched sequences can facilitate the channeling of intermediates between ordinarily nonconsecutive modules. Thus, in addition to substrate-PKS interactions and domain-domain interactions, these short interpolypeptide sequences represent a third determinant of selective chain transfer that must be taken into consideration in the protein engineering of PKSs. Preliminary biophysical studies on synthetic peptide mimics of these linkers suggest that they may adopt coiled-coil conformations.
Although the role of protein-protein interactions in transducing signals within biological systems has been extensively explored, their relevance to the channeling of intermediates in metabolism is not widely appreciated. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two related families of modular megasynthases that channel covalently bound intermediates from one active site to the next. Recent biochemical studies have highlighted the importance of protein-protein interactions in these chain transfer processes. The information available on this subject is reviewed, and its possible mechanistic implications are placed in context by comparisons with selected well-studied multicomponent protein systems.
BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are commonly used antihypertensive medications that have been reported to affect aberrant angiogenesis and the dysregulated inflammatory response. Because of such mechanisms, it was hypothesized that these medications might affect the tumor response to neoadjuvant radiation in patients with rectal cancer. METHODS: One hundred fifteen patients who were treated with neoadjuvant radiation at the University of Wisconsin (UW) between 1999 and 2012 were identified. Univariate analyses were performed with anonymized patient data. In a second independent data set, 186 patients with rectal cancer who were treated with neoadjuvant radiation at the Queen's Medical Center of the University of Hawaii (UH) between 1995 and 2010 were identified. These data were independently analyzed as before. Multivariate analyses were performed with aggregate data. RESULTS: Among patients taking ACEIs/ARBs in the UW data set, a significant 3-fold increase in the rate of pathologic complete response (pCR) to neoadjuvant therapy (52% vs 17%, P 5 .001) was observed. This finding was confirmed in the UH data set, in which a significant 2-fold-increased pCR rate (24% vs 12%, P 5 .03) was observed. Identified patient and treatment characteristics were otherwise balanced between patients taking and not taking ACEIs/ ARBs. No significant effect was observed on pCR rates with other medications, including statins, metformin, and aspirin. Multivariate analyses of aggregate data identified ACEI/ARB use as a strong predictor of pCR (odds ratio, 4.02; 95% confidence interval, 2.06-7.82; P < .001). CONCLUSIONS: The incidental use of ACEIs/ARBs among patients with rectal cancer is associated with a significantly increased rate of pCR after neoadjuvant treatment. Cancer 2016;122:2487-95.
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