Unusual Heme-Binding PAS Domain from YybT Family Proteins▿

YybT family proteins (COG3887) are functionally unknown proteins that are widely distributed among the firmicutes, including the human pathogens Staphylococcus aureus and Listeria monocytogenes. Recent studies suggested that YybT family proteins are crucial for the in vivo survival of bacterial pathogens during host infection. YybT family proteins contain an N-terminal domain that shares minimum sequence homology with Per-ARNT-Sim (PAS) domains. Despite the lack of an apparent residue for heme coordination, the putative PAS domains of BsYybT and GtYybT, two representative members of the YybT family proteins from Bacillus subtilis and Geobacillus thermodenitrificans, respectively, are found to bind b-type heme with 1:1 stoichiometry. Heme binding suppresses the catalytic activity of the DHH/DHHA1 phosphodiesterase domain and the degenerate GGDEF domain. Absorption spectroscopic studies indicate that YybT proteins do not form stable oxyferrous complexes due to the rapid oxidation of the ferrous iron upon O2 binding. The ferrous heme, however, forms a hexacoordinated complex with carbon monoxide (CO) and a pentacoordinated complex with nitric oxide (NO). The coordination of NO, but not CO, to the heme stimulates the phosphodiesterase activity. These results suggest that YybT family proteins function as stress-signaling proteins for monitoring cellular heme or the NO level by using a heme-binding PAS domain that features an unconventional heme coordination environment

Synthesis of (R)-Mellein by a partially reducing iterative polyketide synthase

Mellein and the related 3,4-dihydroisocoumarins are a family of natural products with interesting biological properties. The mechanisms of dihydroisocoumarin biosynthesis remain largely speculative today. Here we report the synthesis of mellein by a partially reducing iterative polyketide synthase (PR-PKS) as a pentaketide product. Remarkably, despite the head-to-tail homology shared with several fungal and bacterial PR-PKSs, the mellein synthase exhibits a distinct keto reduction pattern in the synthesis of the pentaketide. We present evidence to show that the ketoreductase (KR) domain alone is able to recognize and differentiate the polyketide intermediates, which provides a mechanistic explanation for the programmed keto reduction in these PR-PKSs

Insights into the programmed ketoreduction of partially reducing polyketide synthases : stereo- and substrate-specificity of the ketoreductase domain

One of the hallmarks of iterative polyketide synthases (PKSs) is the programming mechanism which is essential for the generation of structurally diverse polyketide products. In partially reducing iterative PKSs (PR-PKSs), the programming mechanism is mainly dictated by the ketoreductase (KR) domain. The KR domain contributes to the programming of PR-PKSs through selective reduction of polyketide intermediates. How the KR domain achieves the selective ketoreduction remains to be fully understood. In this study, we found that the KR domain of the (R)-mellein-synthesizing PR-PKS SACE5532 functions as a B-type KR domain to generate (R)-hydroxyl functionalities. Comparative studies of the KR domains of SACE5532 and NcsB suggested that the two KR domains have distinct substrate preferences towards simple N-acetylcysteamine thioester (SNAC) substrates. We further found that the substrate preference of KRSACE5532 can be switched by swapping several motifs with KRNcsB, and that swapping of the same motifs in the full length SACE5532 resulted in a reprogramming of the PKS. Together, the results advance our understanding of the programming of iterative PR-PKSs by providing new support to the hypothesis that the programmed ketoreduction is accomplished by differential recognition of polyketide intermediates.Accepted versio

Synthesis of (<i>R</i>)-Mellein by a Partially Reducing Iterative Polyketide Synthase

Mellein and the related 3,4-dihydroisocoumarins are a family of natural products with interesting biological properties. The mechanisms of dihydroisocoumarin biosynthesis remain largely speculative today. Here we report the synthesis of mellein by a partially reducing iterative polyketide synthase (PR-PKS) as a pentaketide product. Remarkably, despite the head-to-tail homology shared with several fungal and bacterial PR-PKSs, the mellein synthase exhibits a distinct keto reduction pattern in the synthesis of the pentaketide. We present evidence to show that the ketoreductase (KR) domain alone is able to recognize and differentiate the polyketide intermediates, which provides a mechanistic explanation for the programmed keto reduction in these PR-PKSs