Source: http://www.biochemsoctrans.org/content/46/5/1161
Timestamp: 2019-04-20 00:11:59+00:00

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Flavins are cofactors of many enzymes, mostly of the oxidoreductase class . The most well-known flavins, in order of decreasing occurrence and complexity, are flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin. All three of these flavins contain a tricyclic heterocycle isoalloxazine that is capable of undergoing one-electron or two-electron redox reactions. Most commonly, flavins are noncovalently bound to proteins, but in ∼10% of cases, FAD and FMN form covalent bonds with the carrier proteins . Several types of such bonds, linking the isoalloxazine moiety of FAD or FMN to histidine, tyrosine, or cysteine residues (Figure 1A), have long been known in succinate dehydrogenase, sarcosine oxidase, trimethylamine dehydrogenase, p-cresol methylhydroxylase, and many other enzymes . A different bonding type was identified in Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), in which the phosphate group of FMN forms an ester bond with a threonine residue  (Figure 1B).
Figure 1. Different types of covalent bonds linking flavins to proteins.
(A) Conventional modes of FMN (R=H) and FAD (R=AMP) bonding to the indicated amino acid residues. (B) FMN linked by a phosphoester bond.
Attaching flavin via a covalent bond provides several advantages over noncovalent binding. Linking through the isoalloxazine ring increases the redox potential of the flavin cofactor . FMN that forms a phosphoester bond has a periplasmic location in many proteins [5–9], and the covalent bond prevents cofactor loss to the outer medium. Furthermore, this type of FMN binding should additionally allow mobility of the electron-carrying group, which is deemed to be essential for electron transfer between subunits in complex proteins, such as Na+-NQR .
Although certain flavinylation reactions appear to require a specific chaperone activity , the prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions, and there exists structural evidence in favor of this hypothesis for His-bound FAD in succinate dehydrogenase . However, in 2013, we identified the first flavin transferase that catalyzes flavin transfer from FAD to a threonine residue of Vibrio harveyi Na+-NQR , an important step in Na+-NQR maturation. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Here, we provide the first overview of the progress achieved by studies of this reaction since its discovery.
Na+-NQR is a bacterial respiratory chain-linked membrane protein that oxidizes NADH using ubiquinone as an electron acceptor [14–16]. The Na+-NQR molecule consists of six different subunits (NqrA–F), typically encoded by genes combined into one operon (nqr) [17,18]. The electron transport chain in Na+-NQR is formed by two covalently bound FMN residues (in subunits NqrB and NqrC) [19,20], noncovalently bound FAD and riboflavin molecules [21,22], a [2Fe-2S] cluster [22–24], and a Cys4[Fe] center . Of note, Na+-NQR is the only enzyme that uses riboflavin as a redox cofactor.
The starting point in the discovery of Na+-NQR-specific flavin transferase was the observation that a separate NqrC subunit of Vibrio cholerae Na+-NQR was produced from a plasmid-encoded nqrC gene in a flavinylated form in the host cells, but in a flavin-deficient form in Escherichia coli cells . This observation was later extended by the finding that the E. coli cells transformed with a plasmid containing the complete V. harveyi nqr operon also produced flavin-deficient, nonfunctional Na+-NQR . Since E. coli lacks its own Na+-NQR, a likely corollary was that this bacterium lacks the specific component(s) required for its post-translational modification. Bioinformatics analysis of the genomes of all nqr operon-containing bacteria carried out to pinpoint such component indicated that all such genomes contain an apbE (apb = alternative pyrimidine biosynthesis) gene, located in most cases in the vicinity of the nqr operon . This analysis suggested a functional link between the corresponding protein product, ApbE, and Na+-NQR.
The apbE gene was initially identified among the genes essential for thiamine synthesis. Mutations in apbE resulted in a conditional thiamine auxotrophy in Salmonella enterica . The gene encodes the ∼40 kDa lipoprotein, ApbE, also referred to as Ftp (flavin trafficking protein) bound to the periplasmic side of the inner membrane in S. enterica . The periplasmic location of ApbE suggested that it is only indirectly involved in thiamine synthesis, which is localized to the cytoplasm. The apbE gene was additionally found to be important for [Fe-S] cluster assembly , and this link may explain the apbE requirement for thiamine synthesis, since it depends on a 4-amino-2-methyl-5-hydroxymethylpyrimidine monophosphate synthase (ThiC) that contains an essential oxygen-labile [Fe-S] cluster . Finally, Boyd et al.  demonstrated by X-ray crystallography that S. enterica ApbE can bind FAD.
The association of ApbE with Na+-NQR became evident when it was found that disruption of the apbE gene abolished the quinone reductase activity of Na+-NQR, but did not affect the activities of other respiratory enzymes in Klebsiella pneumoniae . Further in vitro and in vivo experiments confirmed and detailed this association. In both the NqrC and NqrB subunits of V. harveyi Na+-NQR, FMN is bound to a threonine residue found in the same DGxSGAT motif . Co-expression of the V. harveyi nqrC and apbE genes in E. coli resulted in the production of a flavinylated NqrC subunit, which was produced in a flavin-deficient apo-form in the absence of the apbE gene . Furthermore, the apo-form of NqrC could be flavinylated in vitro by incubation with FAD and ApbE . These findings indicated that ApbE is a specific post-translationally modifying enzyme that possesses an FAD:protein FMN transferase activity, according to the IUBMB Enzyme Commission nomenclature. The Commission later issued the number EC 2.7.1.180 to the novel enzyme (BRENDA, The Comprehensive Enzyme Information System, http://www.brenda-enzymes.org/).
ApbE-catalyzed flavin transfer depends on divalent cations and is completely inhibited by EDTA . Mg2+ is the best cofactor; however, Ca2+, Mn2+, Zn2+, Co2+, Ba2+, and Cd2+ can also confer partial activity, depending on the ApbE source [8,33]. Reduced and oxidized forms of FAD are equally effective as FMN donors  and cannot be replaced in this capacity by FMN or riboflavin . The dissociation constant for the ApbE–FAD complex is in the range of 0.4–2.2 μM [8,33,34].
In addition to being able to flavinylate proteins, Treponema pallidum ApbE catalyzes the slow, ‘single-turnover’ hydrolysis of FAD to yield FMN and AMP (FAD pyrophosphatase reaction) . This aberrant activity was detected in only some bacterial ApbEs with weak FAD binding, caused by a Tyr/Asn replacement in the coordination sphere of the isoalloxazine ring , and is outside the scope of this review.
Na+-NQR subunits are not the only protein substrates for ApbE. Many bacteria, including E. coli, and some archaea contain a similar to Na+-NQR ferredoxin:NAD+ oxidoreductase (RNF) complex , which catalyzes electron transfer between ferredoxin and NAD+, coupled with transmembrane Na+ transport . Five of its six subunits are homologous to the Na+-NQR NqrA–E subunits, and two of them (RnfD and RnfG, paralogs of NqrB and NqrC, respectively) contain FMN bound by a phosphoester bond . The RnfG subunit has been directly demonstrated to be flavinylated by ApbE in E. coli . The apbE-deficient strain of S. enterica was unable to produce a functional RNF complex, as indicated by defective [Fe-S] cluster metabolism , which depends on RNF activity . These links further detail the molecular mechanism of the apbE-associated thiamine auxotrophy .
Other bacterial flavoproteins in which FMN is linked by a phosphoester bond include the electron-transferring proteins, NosR and PceC [8,9], the urocanate reductase UrdA , and the cytoplasmic fumarate reductase KPK_2907 . All these are homologs of the Na+-NQR NqrC subunit and, similar to the latter, contain an FMN-binding domain (FMN_bind, PFAM ID: PF04205), which contains a flavin-accepting threonine . These proteins require ApbE for flavinylation [5,8,9,13]; thus, covalent attachment of FMN via a phosphoester bond is a widespread post-translational modification reaction in prokaryotes.
The Protein Data Bank lists 15 X-ray crystal structures of ApbE and its genetically engineered variants from five bacteria: Thermotoga maritima , S. enterica , T. pallidum [34,42], E. coli , and Pseudomonas stutzeri . Depending on the origin of the enzyme, the crystals contain the protein in a monomeric or dimeric form. Each monomer is composed of three domains, two of which adopt a tunneling fold (T-fold) with a ββααββ topology (Figure 2A), and can be superimposed with a root mean square deviation of 1.8–2.4 Å . The third domain, resembling a three-helix bundle fold, is found between the T-fold domains in the protein sequence. In P. stutzeri ApbE, this domain is stabilized by a single disulfide bond not found in other ApbEs.
Figure 2. The structure of T. pallidum ApbE (PDB entry: 4IFX) .
(A) The overall view of the complex with FAD (shown as a spherical representation) and two Mg2+ ions (shown as green spheres). (B) Coordination of Mg2+ in the bi-metal center. Created with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4, Schrödinger, LLC).
The flavin and related ligands found in the structures include FAD (PDB ID: 3PND , 4IFX , and 4XDT ), ADP (4IFZ, 4IG1 , and 4XGX ), and AMP (4IFZ and 4IG1 ), and are all found at the interface of the three domains. The FAD molecule adopts an unusual bent conformation not found in other FAD-binding proteins. As a result, the isoalloxazine and adenine rings, in particular the latter, are submerged into the protein molecule and are not easily accessible to solvent. The pyrophosphate moiety is more exposed to the medium and accessible to attack by the threonine hydroxyl of the target protein. In their complexes, the AMP and ADP molecules occupy the same position as the corresponding portions of the FAD molecule. The isoalloxazine ring is sandwiched between the Tyr78 and Met41 side chains in S. enterica ApbE  and between Asn55 and Ile17 in T. pallidum ApbE  (see Supplementary Figure S1), the latter of which is a less stabilizing interaction than the π-stacking of Tyr78. The hydrogen-bonding interactions that stabilize adenine occur predominantly with the protein backbone.
Consistent with the requirement of the flavinylation reaction for divalent metal ions, the T. pallidum ApbE–FAD complex contains two Mg2+ ions (Mg1 and Mg2) that form a bi-metal center , a presumable core of the catalytic site (Figure 2B). Both Mg2+ ions are co-ordinated to the Asp284 and AMP phosphate oxygens. Mg1 is further co-ordinated to the Ala162 and Asp284 carbonyls and the Thr288 hydroxyl, whereas Mg2 interacts with the oxygens from both phosphate groups of the FAD molecule. Other metal ligands are water molecules. Asp284 and Thr288 are conserved in all ApbEs (Supplementary Figure S1), and the Asp284Ala replacement renders ApbE inactive , confirming the importance of the divalent cations for the flavin transferase activity. The coordination of the two Mg2+ ions in the complex with the inhibitor ADP was only slightly changed . In the complex with the product, AMP, a water molecule, replaces the second phosphate oxygen as the Mg2 ligand. The distance between the two Mg2+ ions is ∼3.2 Å in all structures. ApbE crystallized in the absence of FAD contained only one Mg2+ ion found in the Mg1 site, where the Thr288 ligand was replaced by a water molecule .
Phosphoryl-transfer reactions are typically assisted by a general acid to stabilize the leaving group and by a general base to activate the nucleophile. The FAD-containing structures revealed a protonated histidine residue (His256 in T. pallidum ApbE) hydrogen-bonded to the oxygen atom that bridges two FAD phosphates [32,34,42]. This residue is absolutely conserved in the known ApbE proteins (Supplementary Figure S1), and its mutations inactivate ApbE without affecting FAD binding [33,42]. The histidine residue is therefore the most likely candidate for the general acid that protonates the AMP phosphate. The activation of the nucleophilic OH-group of threonine is apparently achieved by its interaction with Lys211 (V. harveyi NqrC numbering) in the target protein . This lysine residue is conserved in all NqrC sequences, points directly at the acceptor threonine residue in several NqrC structures [25,35,43], and its mutation prevents the flavinylation reaction . Kinetic studies have indicated that a deprotonated group with a pKa of 8.4 is required for catalysis by V. cholerae ApbE . This value is two pH units less than that of a free amino group, consistent with it being hydrogen-bonded to the threonine hydroxyl in the protein substrate.
The ApbE–FAD complex is therefore well suited to attack by the hydroxyl group of the target protein. The bi-metal center found in ApbE and its coordination to the FAD phosphates are similar to those found in other phosphoryl-transfer enzymes . By coordinating the phosphate oxygens, the center assists in positioning the substrate, transition state, and products, neutralizing their negative charge . Furthermore, two amino acid residues, one in ApbE and the other in the target protein, may assist catalysis by acting as a general acid and base, respectively. Importantly, point mutations in the catalytic machinery cancel the flavinylation reaction without affecting FAD binding; thus, ApbE satisfies the criteria needed to classify this protein as an enzyme with a substrate-assisted catalysis, rather than simply a chaperone. In fact, ApbE combines both enzymatic and chaperone-like properties; while primarily being a catalyst, ApbE exhibits high affinity for FAD, thereby directing the cofactor to its target protein. The chaperone function of ApbE is of particular importance in the modification of periplasmically localized proteins, since it prevents the loss of FAD, presumably delivered from the cytoplasm by MATE (multidrug and toxic compound extrusion) protein family transporters [46,47].
There exists limited information regarding the kinetic parameters of the ApbE-catalyzed reaction. The catalytic constant can be roughly estimated to be less than 1 min−1 [33,42]. The Km value for FAD is less than 1 µM; such that the enzyme is almost saturated at an FAD concentration of 1 µM . In contrast, the Km value for the protein substrate is relatively high, and the reaction rate shows a linear dependence on [NqrC] up to a concentration of 100 µM . At a saturating FAD concentration (1 mM), the effect of 1 mM AMP, ATP, or FMN was small, if any [33,35,42]. Surprisingly, 1 and 10 mM ADP activated ApbE ∼6-fold, despite the fact that ADP binds in the same site as FAD. K+ (100 mM), but not Na+, exerted a 10-fold activation, which was Na+-independent ; however, the mechanisms of these effects are unknown.
A remark on the activity assay is warranted at this point. In vitro measurements of the flavinylation reaction make use of soluble truncated forms of ApbE and NqrC, without their membrane-embedded segments. The reaction is carried out in solution and quantitated by measuring the fluorescence of the flavinylated NqrC band following SDS–PAGE electrophoresis of the reaction medium. The above-mentioned kinetic parameters, which were estimated in this way, may therefore differ from those for the full-size membrane-bound proteins reacting in vivo.
The above description refers to flavin transferase as a separate entity; nevertheless, recently published evidence suggests that proteins can be flavinylated intramolecularly — by an ApbE-like domain present in the target protein. Kinetoplastid Leptomonas pyrrhocoris NADH:fumarate oxidoreductase (FRD) is the only such protein described to date . The FRD molecule is formed by a single polypeptide that is organized into two catalytic domains and an additional N-terminal domain that is homologous to the prokaryotic ApbE proteins . When the L. pyrrhocoris FRD gene was expressed in yeast cells, a fully functional FRD was produced, which contained a covalently bound FMN residue in the N-terminal extension that did not belong to any domain . The flavin acceptor in FRD was a serine found in the same sequence motif as the acceptor threonine in the above-described proteins. This post-translational modification is important for FRD function, since replacement of the acceptor serine abolished both the flavinylation and the catalytic activity of FRD . Noteworthy, L. pyrrhocoris does not contain any homologs of the Na+-NQR NqrB and NqrC subunits that could serve as substrates for this putative flavin transferase; and the yeast genome does not contain apbE genes to produce its own flavin transferase that could flavinylate the L. pyrrhocoris FRD. These findings therefore suggested that FRD was flavinylated by its own inbuilt flavin transferase domain. The ApbE domain and flavinylation motif are conserved in FRDs from other kinetoplastids including important pathogens (Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, and others) , suggesting a similar autoflavinylation modification.
E. coli cells contain a single apbE gene, and its product flavinylates only the host RNF subunits, but not other homologous proteins heterologously produced in E. coli, such as NqrC from V. harveyi , V. cholerae , or Shewanella oneidensis , urocanate reductase from S. oneidensis , fumarate reductase KPK_2907 from K. pneumoniae , the electron-transferring protein NosR from P. stutzeri , or PceC from Desulfitobacterium hafniense . Moreover, there are no indications that this flavin transferase modifies any protein other than RNF in the host cell. Thus, E. coli ApbE is currently an example of an ‘absolutely specific’ flavin transferase, since it modifies no other protein besides its host RNF (though at two sites). Similarly, the T. pallidum TP0171 lipoprotein is flavinylated by its host ApbE, but not by Treponema denticola ApbE . The ApbE-like domain of the autoflavinylated FRD apparently represents an extreme case of absolute specificity. The presence of up to five different apbE genes in certain bacteria  suggests that their product flavin transferases are also specific enzymes and recognize only one target in the whole spectrum of cellular proteins to be flavinylated.
However, examples of interspecies cross-reactivity have also been documented; V. cholerae ApbE modified S. oneidensis urocanate reductase  and K. pneumoniae fumarate reductase  in addition to the host Na+-NQR . Consistent with the low specificity of V. cholerae ApbE, the genome of this bacterium contains a single apbE gene but four genes of potential flavinylation targets, bearing the flavinylation motif . Similarly, T. pallidum ApbE flavinylated T. denticola NqrC , and Marinobacter hydrocarbonoclasticus ApbE was active against the P. stutzeri NosR protein . At least in the last two cases, the interspecies modification reaction proceeded much slower than with the host targets.
Evidently, the specificity of a particular flavin transferase is governed by both its active site structure and the recognition motif, together with its structural context in the target protein. The recognition motif is highly degenerate in the target proteins, which may contribute to specificity. The consensus flavinylation motif can be written as DgxtsAT/S, with the acceptor residue shown in bold. However, neither residue seems to be invariant in all the sequences in which the flavinylation was experimentally measured (Figure 3) or predicted. As mentioned above, the FMN-accepting amino acid residue in the target protein is either a threonine or a serine. The first residue has naturally replaced an asparagine in T. denticola NqrC , and an artificial Asp-to-Ala replacement in S. oneidensis NqrC had no effect on its flavinylation . Furthermore, the aspartic acid residue is replaced by glutamic acid or an even positively charged residue in the FMN_bind domain (PF04205) of several bacterial protein sequences found in GenBank, although it is unknown whether the corresponding motifs are indeed flavinylated.
Figure 3. Partial sequence alignment of all proven ApbE targets containing the flavinylation motif (underlined).
The position of the FMN acceptor residue is marked by an asterisk. Vh_NqrC, Vc_NqrC, So_NqrC, and Td_NqrC, the Na+-NQR NqrC subunit from V. harveyi, V. cholerae, S. oneidensis, and T. denticola (accession numbers Q9RFV9.4, AKB08163.1, AAN54176.1, and AAS11327.1, respectively); Vc_RnfG and Ec_RnfG, the RNF RnfG subunit from V. cholerae and E. coli (ACP09040.1 and ART24401.1, respectively); So_UrdA, S. oneidensis urocanate reductase (Q8CVD0.1); Ps_NosR, the P. stutzeri electron-transferring NosR protein (CAA78380.1); Dh_PceC, the D. hafniense electron-transferring PceC protein (CAG70351.1); Kp_2907 fumarate reductase from K. pneumoniae (KPK_2907); Tp_0171, the T. pallidum periplasmic lipoprotein (P16055.1); and Lp_gFRD, L. pyrrhocoris glycosomal FRD (ABB37_00293).
At least one type of covalent flavin attachment to proteins requires the specific flavin transferase enzyme, ApbE, which is widely distributed among living organisms. GenBank lists ∼96 000 bacterial, 236 archaeal, and 119 eukaryotic ApbE-like sequences. In bacteria, apbE genes are distributed among many taxa. Most archaeal apbE sequences belong to methanogenic organisms, which contain an RNF analog, ferredoxin:methanophenazine oxidoreductase complex , and most eukaryotic sequences belong to Kinetoplastida.
An important outcome of the bioinformatics analysis is that the apbE gene has a much wider distribution among different bacterial taxa than the genes for the known ApbE targets. For instance, the genomes of many Bacilli, Actinobacteria, Aquificae, Chloroflexi, Deinococcus-Thermus, Fibrobacteres-Acidobacteria, and Halobacteriaceae contain apbE-like genes, but no nqr or rnf genes. A likely corollary is that the corresponding organisms possess yet unknown proteins that undergo ApbE-mediated covalent flavinylation. Future studies will identify these proteins and reveal the role of the specific ApbE flavin transferases in their maturation.
Flavin transferase and its gene may find important applications in practice. Genetic constructs bearing genes encoding both ApbE and a target protein have been suggested as tools for localization-dependent fluorogenic protein modification in bacterial and eukaryotic cells [5,52]. The apbE gene can also be used in synthetic biology to produce flavoproteins. Finally, ApbE inhibitors may help to selectively combat various infectious diseases caused by Na+-NQR-, RNF- or FRD-containing microorganisms.
We gratefully acknowledge support from the Russian Science Foundation [research project 14-14-00128].

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