Identifying the Mechanism of Reduced Flavin Transfer in Alkanesulfonate Monooxygenase System

Abstract

The two‐component alkanesulfonate monooxygenase system from bacteria are involved in the acquisition of sulfonates during sulfurlimiting conditions. In this two‐component system the FMN reductase (SsuE) provides reduced flavin to the monooxygenase enzyme (SsuD). The monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of the carbon‐sulfur bond of alkanesulfonates, leading to the formation of the corresponding aldehyde and sulfite product.The transfer of reduced flavin from SsuE to SsuD may involve either a channeling or dissociative mechanism. Prior studies have identified stable protein‐protein interactions between SsuE and SsuD supporting a channeling mechanism for flavin transfer. The SsuD enzyme belongs to the bacterial luciferase family that includes bacterial luciferase and LadA. Members of this family have a characteristic TIM‐barrel conformation with several insertion regions. The mobile loop contained on the insertion region was shown to be involved in stabilizing loop closure to protect reaction flavin intermediates, but the role of this region in flavin transfer has not been evaluated. HDX‐MS studies were conducted to identify potential interaction sites on each enzyme. The SsuD protected sites were located on the mobile loop region, while the protected sites on SsuE were near the putative active site region. There are noticeable charged and polar residues in these regions, which may play a role in the formation of a transient stable‐structure. Variants of these polar regions were generated to evaluate protein‐protein interactions and flavin transfer were altered. The interaction sites on SsuE and SsuD have the potential to act as a dynamic region which enables the transfer of reduced flavin between the two proteins.