Flavoenzymes: Covalent versus Noncovalent

Abstract

The use of nonprotein cofactors by enzymes expands the range of biological chemistries supported in nature. Flavins, which are derivatives of vitamin B2, 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‚Äźassociated 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.

Keywords: flavoenzymes; flavin; covalent flavinylation; flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); cofactors

Figure 1. (a) Chemical structure of riboflavin with the isoalloxazine labelled in a manner consistent with this text. An equivalent, alternative labelling scheme terms the ‘8α’ group as ‘C8M’, and this nomenclature may be seen in other publications. The flavin mononucleotide and flavin adenine dinucleotide additions to the ribityl side chain are shown. (b) Chemical structures of the amino acids that are known to form a covalent bond with flavin. The associated ring structures are numbered. An equivalent, alternative nomenclature for the nitrogens of the histidine imidazole ring terms N(1) as Nδ and N(3) as Nϵ. Accordingly, 8α‐N(3)‐histidyl linkage and C8M‐Nϵ‐histidyl linkage are equivalent terms. For all structures, potential sites of covalent linkage are colored in red.
Figure 2. A range of physiological processes catalysed by flavoenzymes.
Figure 3. (a,b) Comparison of the FAD of (a) human monoamine oxidase A [2.2 Å, PDB 2Z5X, teal (Son et al., )], which contains an FAD covalently attached to Cys406 with (b) putrescine oxidase from R. erythropolis [2.3 Å, PDB 2YG4 (Kopacz et al., )] with electron density is shown as (2|Fo| − |Fc|) maps contoured at 2σ around the FAD. Key residues for cofactor binding are highlighted as sticks.
Figure 4. Ancillary proteins that promote covaletn flavinylation. (a) Space filling model of SdhE [PDB 1X6J, blue (Lim et al., )] with the most highly conserved residues shown in gold. (b) Ribbons model of SdhE shown in the same orientation as panel, with the RGxxE motif highlighted (a). (c) Alignment of the structures of p‐cresol methylhydroxylase (Cunane et al., ) with (PDB 1WVE, green) and without the cyctochrome subunit (PDB 1WVF, purple) identifies slight conformational changes in the flavoprotein upon cytochrome association.
Figure 5. Structure of the flavin transferase, ApbE [PDB 3PND (Boyd et al., )]. Noncovalent FAD is shown in yellow. The inset highlights the buried exposure of the isoalloxazine ring, which may assist in flavin transfer to the flavoenzyme.
Figure 6. Quinone methide mechanism for attachment at the 8α carbon of the flavin. Blue ‘B’s represent catalytic bases, which are likely amino acid side chains. The red ‘AA’ indicates the amino acid that is covalently attached.
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Starbird, Chrystal A, Maklashina, Elena, Cecchini, Gary, and Iverson, TM(Sep 2015) Flavoenzymes: Covalent versus Noncovalent. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0026073]