Covalent flavinylation of vanillyl‐alcohol oxidase is an autocatalytic process

Abstract

For most reported flavoproteins, the flavin cofactor is noncovalently but tightly bound by noncovalent interactions [1]. Nevertheless, a small but significant group of flavoproteins ( 5%) contains a covalently bound flavin. In most of these so-called covalent flavoproteins, the flavin cofactor is attached to the protein at the 8a-methyl of the isoalloxazine moiety, whereas some C6-linked flavins also have been found [2]. The most common linkage type involves coupling to a histidine residue, but proteins containing cysteinyl and tyrosyl linked flavins have also been reported. Recently, some covalent flavoproteins were even found to harbour a FAD cofactor that is tethered via two covalent linkages: a 8a-histidyl-C6-cysteinyl bound FAD [3]. The mechanism by which flavin cofactors are covalently incorporated is largely unknown, as is the rationale for covalent histidyl-flavin attachment. Previous studies have hinted at an autocatalytic process in which no helper enzymes or other additional factors are needed [2,[4][5][6]. This is in contrast to many other Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8a-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K FAD = 2.3 lm, k activation = 0.13 min )1

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

“…Using the riboflavin auxotrophic E. coli strain BSV11, we obtained an apoprotein preparation that contained 17% residual activity. Spectral analysis confirmed the presence of some holoenzyme, analogous to observations made with sarcosine oxidase and vanillyl-alcohol oxidase2829. More striking, the obtained apoenzyme can be fully reconstituted with either FMN or FAD, leading to quite identical catalytic properties.…”

“…Using riboflavin auxotrophic strains, both flavoenzymes that bind their flavin covalently or non-covalently can be produced in their apo-form. Examples include sarcosine oxidase28 and vanillyl-alcohol oxidase29.…”

Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

“…In the case of SdhCDAB, it was recently discovered that the SdhE chaperone directly interacts with the SdhA subunit to mediate flavinylation (32, 48), and the absence of the equivalent SdhAF2 chaperone in humans results in paraganglioma tumorigenesis (33). Following insertion of FAD into its binding pocket and repositioning of the capping domain, covalent attachment is thought to be an autocatalytic process, a common theme that is conserved in other flavoenzymes (49–51). In this study, we show the SdhA-R286 residue to be absolutely essential for flavinylation.…”

Communication. Vanillyl alcohol oxidases produced in Komagataella phaffii contain a highly stable noncovalently bound anionic FAD semiquinone

“…VAO covalently binds its FAD cofactor via His422 [5][6][7]. The proposed sequence of events of this posttranslational modification of VAO is as follows: Before any incorporation of FAD can occur, the VAO-polypeptide folds and oligomerises to the dimer.…”

“…Covalent incorporation increases the redox potential of the flavin cofactor and stimulates efficient redox catalysis. The full process takes approximately 30-60 minutes [5][6][7].…”