Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.

Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: a rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins.

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of samples of the native and recombinant wild-type trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in trimethylamine dehydrogenase that involves an electrophilic flavin iminoquinone methide. The proposed mechanism involving the flavin iminoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enzymes via the 8alpha position, thus providing a rationale for the evolution of covalent flavoproteins in general. Covalent linkage at C6 or the 8alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone methide, respectively.

Selective modification of alkylammonium ion specificity in trimethylamine dehydrogenase by the rational engineering of cation-pi bonding.

In trimethylamine dehydrogenase (TMADH), substrate is bound in the active site by organic cation-pi bonding mediated by residues Tyr-60, Trp-264, and Trp-355. In the closely related dimethylamine dehydrogenase (DMADH), modeling suggests that a mixture of cation-pi bonding and conventional hydrogen bonding is responsible for binding dimethylamine. The active sites of both enzymes are highly conserved, but three changes in amino acid identity (residues Tyr-60 --> Gln, Ser-74 --> Thr, and Trp-105 --> Phe, TMADH numbering) were identified as probable determinants for tertiary --> secondary alkylammonium ion specificity. In an attempt to switch the substrate specificity of TMADH so that the enzyme operates more efficiently with dimethylamine, three mutant proteins of TMADH were isolated. The mutant forms contained either a single mutation (Y60Q), double mutation (Y60Q x S74T) or triple mutation (Y60Q x S74T x W105F). A kinetic analysis in the steady state with trimethylamine and dimethylamine as substrate indicated that the specificity of the triple mutant was switched approximately 90,000-fold in favor of dimethylamine. The major component of this switch in specificity is a selective impairment of the catalytic efficiency of the enzyme with trimethylamine. Rapid-scanning and single wavelength stopped-flow spectroscopic studies revealed that the major effects of the mutations are on the rate of flavin reduction and the dissociation constant for substrate when trimethylamine is used as substrate. With dimethylamine as substrate, the rate constants for flavin reduction and the dissociation constants for substrate are not substantially affected in the mutant enzymes compared with wild-type TMADH. The results indicate a selective modification of the substrate-binding site in TMADH (that impairs catalysis with trimethylamine but not with dimethylamine) is responsible for the switch in substrate specificity displayed by the mutant enzymes.

Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine.

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

The flavinylation reaction of trimethylamine dehydrogenase. Analysis by directed mutagenesis and electrospray mass spectrometry.

The flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from Methylophilus methylotrophus (bacterium W3A1) and Escherichia coli were studied by electrospray mass spectrometry (ESMS). The ESMS analyses demonstrated for the first time that wild-type enzyme expressed in M. methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme. ESMS demonstrated that the deflavo forms of the recombinant wild-type and mutant enzymes are not post-translationally modified and therefore prevented from assembling with flavin mononucleotide (FMN) because of previously unrecognized modifications. The data suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expression levels in E. coli. Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative proportion of flavinylated product was substantially less than that seen for the recombinant wild-type enzyme. No flavinylation products were observed for a double mutant (His-29 to Cys-29; Cys-30 to His-30), in which the positions of the putative flavinylation base and cysteine nucleophile were exchanged. Taken together, the data indicate that the assembly of trimethylamine dehydrogenase with FMN occurs during the folding of the enzyme, and in the fully folded form, deflavo enzyme is unable to recognize FMN. Results of site-directed mutagenesis experiments in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced. Consequently, in the folded mutant enzymes, less flavin is trapped in the active site, and reduced levels of flavinylated product are obtained.