Molecular insights into the enzymatic diversity of flavin‐trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm

Abstract

We recently reported a flavin‐trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal‐dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal‐dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal‐dependent Ftp catalysis in either Nqr‐ or Rnf‐redox‐containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD‐binding protein to a Mg2+‐dependent FAD pyrophosphatase (Ftp_Tp‐like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox‐carrying proteins (e.g., RnfG_Ec) via the metal‐dependent covalent attachment of FMN. A high‐resolution structure of the Ftp‐mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester‐threonyl‐FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated.

Kinetic characterization of Vibrio cholerae ApbE: Substrate specificity and regulatory mechanisms

“…The UV- vis spectrum of the purified sample shows the typical flavin absorption peaks at 360 and 450 nm ( Fig 1C left panel dashed line), corresponding to a FAD: protein ratio of 0.24±0.01 ( Fig 1D ). The relatively low content of flavin compared to other ApbE preparations [ 37 , 42 ] can be explained by the substantial purification procedure carried out to obtain a highly pure sample. In other members of the family FAD tends to dissociate slowly during protein purification [ 37 , 42 ].…”

“…The relatively low content of flavin compared to other ApbE preparations [ 37 , 42 ] can be explained by the substantial purification procedure carried out to obtain a highly pure sample. In other members of the family FAD tends to dissociate slowly during protein purification [ 37 , 42 ]. To corroborate that ApbE is purified in its active flavin-binding form, the sample was incubated with a molar excess of FAD (50:1) for 1 h, and washed thoroughly by four rounds of dilution (1:20) and re-concentration.…”

Biophysical insights into a highly selective L-arginine-binding lipoprotein of a pathogenic treponeme

“…The second mutation was changing to alanine the glutamate whose side chain made a presumed salt bridge with the guanidinium moiety of the bound amino acid (E128). This mutation was more deleterious to Larginine binding than the previous, displaying a KD that was higher than the wild-type by approximately 5,000X (31 [26,37] μM). These results allowed us to conclude that the binding site observed in the crystal structure is the operative one in solution, and that the abrogation of a single salt bridge, while not enough to fully eliminate binding, has significantly negative effects on the binding of L-arginine to Tv2483.…”

“…The cells were then induced for ~20 h with 0.2% (w/v) Larabinose at 16 °C and harvested, and cell pellets were stored at -80 °C. The procedures for expression and purification of the recombinant proteins were essentially as previously described (36,37).…”

Functional clues from the crystal structure of an orphan periplasmic ligand‐binding protein from Treponema pallidum

“…The culture was then induced for 3 h at 37°C with 0.2% (w/v) L‐arabinose. The procedures for expression and purification of the recombinant proteins were essentially as previously described …”