Abstract The use of nonprotein cofactors by enzymes expands the range of biological chemistries supported in nature. Flavins, which are derivatives of vitamin B 2 , are highly conjugated rings that are particularly useful for oxidoreduction and group transfer reactions. Most flavins are noncovalently associated with their enzymes, but around 10% of flavoproteins have the flavin covalently attached in vivo . Extensive research has investigated how the presence of the covalent bond between enzyme and flavin cofactor influences enzymatic catalysis. This work identified that the primary roles of the covalent flavin are to allow catalysis of more thermodynamically challenging reactions and to prevent the cofactor from disassociating from the enzyme. Major questions in the field now include the mechanism of covalent flavinylation. The earliest studies on a subset of covalent flavoproteins suggested that cofactor attachment could be an autocatalytic posttranslational process. However, the recent identification of assembly factors that promote covalent flavinylation identifies that ancillary proteins may be important for covalent flavinylation in vivo . Key Concepts Covalent flavin attachment increases stability of the holoenzyme and increases the enzyme's redox potential. Covalent flavinylation may occur either through an entirely autocatalytic mechanism, or be assisted by assembly factors. Enzyme‐associated flavin can promote a variety of chemistries. Flavoenzymes can have covalent or noncovalent flavin. Covalent flavinylation can occur on multiple sites of the protein and flavin molecule.
The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely of repeats of the FliF subunit. This MS-ring is critical for the assembly and stability of the entire flagellum. Despite multiple independent cryoEM structures of the MS-ring, there remains a debate about the stoichiometry and organization of the ring-building motifs (RBMs). Here, we report the cryoEM structure of a Salmonella MS-ring that was first assembled into the flagellar switch, then separated from this larger complex. Using 2D class averages, we show that the post-assembly MS-ring can contain 32, 33, or 34 FliF subunits, with 33 being the most common. RBM3 has a single location with C32, C33, or C34 symmetry. RBM2 is found in two locations with RBM2inner having C21 or C22 symmetry and an RBM2outer-RBM1 having C11 symmetry. Comparison to previously reported structures identifies several differences. Most strikingly, we find that the membrane domain forms 11 regions of discrete density at the base of the structure rather than a contiguous ring, although density could not be unambiguously interpreted. We further find density in some previously unresolved areas, and we assigned amino acids to those regions. Finally, we find differences in interdomain angles in RBM3 that affect the diameter of the ring. Together, these investigations support a model of the flagellum with structural plasticity, which may be important for flagellar assembly and function.
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