N5 Is the New C4a: Biochemical Functionalization of Reduced Flavins at the N5 Position

Abstract

For three decades the C4a-position of reduced flavins was the known site for covalency within flavoenzymes. The reactivity of this position of the reduced isoalloxazine ring with the dioxygen ground-state triplet established the C4a as a site capable of one-electron chemistry. Within the last two decades new types of reduced flavin reactivity have been documented. These studies reveal that the N5 position is also a protean site of reactivity, that is capable of nucleophilic attack to form covalent bonds with substrates. In addition, though the precise mechanism of dioxygen reactivity is yet to be definitively demonstrated, it is clear that the N5 position is directly involved in substrate oxygenation in some enzymes. In this review we document the lineage of discoveries that identified five unique modes of N5 reactivity that collectively illustrate the versatility of this position of the reduced isoalloxazine ring.

Keywords: C4a-position; N5-position; covalent; flavin; imino; oxide; peroxo.

FIGURE 1

Examples of catalytically relevant flavin-N5 adducts.

FIGURE 2

Overview of the proposed reaction of UbiX. UbiD, Pad1, and Fdc1.

FIGURE 3

A hypothetical chemical mechanism for UbiX/Pad1 and supporting crystal structures of UbiX_ox⋅DMP (dimethylallyl monophosphate) complex (PDB code 4ZAF, green), UbiX_red⋅IMP (isopentyl monophosphate) complex (PDB code 6QLH, blue), UbiX-dimethylallyl monophosphate adduct (PDB code 4ZAV, tan), and UbiX⋅FMN_pren complex (PDB code 4ZAW, yellow).

FIGURE 4

Proposed catalytic mechanism of Fdc1_UbiX and supporting crystal structures of prFMN_ketamine⋅α-FCA (α-fluoro cinnamic acid) (PDB code 4ZAB, blue), prFMNradical (PDB code 6R2T, green), prFMNiminium⋅α-FCA complex (PDB code 4ZAB, light blue), prFMN_int1 adduct (PDB code 6R3O, black), prFMN_int2 adduct (PDB code 6R3F, tan), prFMN_int3 adduct (PDB code 6R3J, purple), and prFMNiminium⋅4-VG (4-vinyl glycol) complex (PDB code 4ZAA, yellow).

FIGURE 5

Reaction catalyzed by UDP-galactopyranose mutase.

FIGURE 6

The active site substrate contacts observed in the structure of the A. fumigatus, UGMox⋅NADPH complex (PBD Code 4VWT, light blue) UGMox⋅UDP-galp complex (PDB Code 3UKH, gray), H63A variant UGMox-galpoUDP complex (PDB Code 5HHF, green), and UGMredoUDP complex (PDB Code 3UTG, purple).

FIGURE 7

The reaction catalyzed by EncM and subsequent conversion to enterocin by EncK and EncR.

FIGURE 8

The Active Site of EncM. Left. EncM with a hydroxytriketide substrate analog bound (PDB code 3W8Z). Right top, EncM crystalized in the presence of 15 bar dioxygen (PDB code 6FOQ). Right middle, EncM in complex with a hydroxytriketide substrate analog bound (PDB code 3W8Z). Right bottom, overlay of both structures. The mesh indicates the shape of the terminal end of the internal cavity. Electron density is shown in blue and derived from 2fo-fc maps.

FIGURE 9

Proposed pathways for the formation of the flavin N5-oxide.

FIGURE 10

Hypothetical chemical mechanism of EncM.

FIGURE 11

Reactions of N5-mediated monooxygenation enzymes; RutA, DszA, and HcbA1.

FIGURE 12

Proposed chemical mechanisms of RutA. Blue, initial mechanism utilizing a C4a-hydroperoxyl. Green, mechanism terminating in the formation of an N5-oxide complex. Red, direct oxygen transfer mechanism utilizing an N5-hydroperoxide and terminating with the formation of an N5-oxide complex.

FIGURE 13

The active site of RutA. Left and right top, RutA in complex with uracil substrate (PDB code 5WAN). For both, active site residues that are involved in N5-oxo formation are shown in green. Right bottom, active site of RutA in complex with uracil under 15 bar dioxygen (PDB code 6TEG). Electron density (2fo-fc) is shown in blue mesh and the active site cavity is shown in gray mesh.

FIGURE 14

Summary of the proposed pathways for covalent catalysis of reduced flavin in enzymes.