| Polyketide synthases (PKSs) are a class of multifunctional enzymes that generate remarkable structural complexity and diversity by harnessing a limited repertoire of catalytic functions in a highly controlled fashion. By varying the building blocks used at every stage of chain elongation, or the extent to which the β-ketone functionality is reduced, a great diversity of polyketide natural products are produced, with a strong track record for pharmaceutical use. The key domain in these modular proteins responsible for building block selection is the acyl transferase (AT) domain. Engineering of polyketide synthases has proved fruitful—replacing an AT domain with a homologous AT domain specific for an alternative building block produces a predictably altered polyketide product. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. In our present work, we describe a generally applicable strategy for the overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (∼60 kDa). Briefly, we co-expressed AT domains with the loading acyl carrier protein of the 6-deoxyerythronolide B synthase (DEBS), producing soluble proteins that could be purified to homogeneity. Using this method, we expressed methylmalonyl-CoA specific AT-2 and AT-6 of the DEBS, malonyl-CoA specific AT-2 of the rapamycin synthase (RAPS), and ethylmalonyl-CoA specific AT-5 of the tylactone synthase (TYLS). We developed two assays for measuring the intrinsic specificity of these AT domains. The first utilizes acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor. Steady-state kinetic analysis of this reaction directly probes the didomain specificity. For less active didomains, we can measure the hydrolysis rates of different acyl-CoA substrates, thereby indirectly revealing substrate preferences. We found the specificity of AT domains to be diverse. The DEBS loading didomain accepted a variety of non-carboxylated substrates, such as propionyl-, acetyl- and butyryl-CoA. On the other hand, DEBS AT-2 and AT-6, as well as RAPS AT-2, only utilized their natural substrates. Finally, TYLS AT-2 accepted ethylmalonyl-, propylmalonyl-, and allylmalonyl-CoA, but not malonyl-, methylmalonyl-, dimethylmalonyl-, or isopropylmalonyl-CoA. These studies lay the foundation for further characterization of AT domains. |
