Solution Structures and Backbone Dynamics of a Flavodoxin MioC from Escherichia coli in both Apo- and Holo-forms

Abstract

Flavodoxins play central roles in the electron transfer involving various biological processes in microorganisms. The mioC gene of Escherichia coli encodes a 16-kDa flavodoxin and locates next to the chromosomal replication initiation origin (oriC). Extensive researches have been carried out to investigate the relationship between mioC transcription and replication initiation. Recently, the MioC protein was proposed to be essential for the biotin synthase activity in vitro. Nevertheless, the exact role of MioC in biotin synthesis and its physiological function in vivo remain elusive. In order to understand the molecular basis of the biological functions of MioC and the cofactor-binding mechanisms of flavodoxins, we have determined the solution structures of both the apo-and holo-forms of E. coli MioC protein at high resolution by nuclear magnetic resonance spectroscopy. The overall structures of both forms consist of an ␣/␤ sandwich, which highly resembles the classical flavodoxin fold. However, significant diversities are observed between the two forms, especially the stabilization of the FMN-binding loops and the notable extension of secondary structures upon FMN binding. Structural comparison reveals fewer negative charged and aromatic residues near the FMN-binding site of MioC, as compared with that of flavodoxin 1 from E. coli, which may affect both the redox potentials and the redox partner interactions. Furthermore, the backbone dynamics studies reveal the conformational flexibility at different time scales for both apo-and holo-forms of MioC, which may play important roles for cofactor binding and electron transfer.Flavodoxins are small FMN-binding proteins found mainly in prokaryotes that are involved in the electron transfer chain reactions of various biological processes, including photosynthesis (1, 2), nitrate reduction (3), methionine synthesis (4 -7), biotin synthesis (8 -11), and activation of certain enzymes, such as pyruvate-formate lyase (13) and (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate synthase (14). It has been established by potentiometry that electrons flow from NADPH to flavodoxin reductase and then to flavodoxin, which subsequently transfers the electron to the downstream targets (15). Eukaryotes and most prokaryotes also contain proteins carrying domains homologous to flavodoxins and flavodoxin reductase in a single polypeptide chain, such as the NADPH-cytochrome P450 oxidoreductase (CPR) 2 (16, 17), nitric-oxide synthase (NOS) (18), methionine synthase reductase (19), and sulfite reductase (20,21). These domains function in a similar fashion as the flavodoxins and flavodoxin reductase proteins in prokaryotic cells. Extensive efforts have been devoted to understanding the cofactor binding, redox partner interaction, and electron transfer mechanisms of flavodoxins and flavodoxinlike domains as well as the folding and stability of these proteins (22-26). However, the detailed mechanisms are not clear yet and remain to be further elucidated.Flavodoxins are classified into the long-...

Conformational Dynamics of Escherichia coli Flavodoxins in Apo- and Holo-States by Solution NMR Spectroscopy

“…The structure is generally similar to the previously reported crystal structure of holo-FldA [17], showing a backbone r.m.s.d value of 1.74 Å. It consists a typical long-chain flavodoxin fold, comprising a central five-strand parallel β-sheet (β1: Thr4-Phe8, β2: Ala-31-Asp35, β3: Ile49-Gly53, β4: Leu82-Gly87, β5: Thr115-Val117 and Leu142-Ala143) flanked by five α-helices (α1: Asn14-Leu26, α2: Lys41-Ala46, α3: Cys64-Leu73, α4: Ala101-Ile109, α5: Thr153-Glu167) on two sides.…”

“…We compared the HSQC spectrum of the directly purified YqcA sample with those of the apo- and holo-forms, and confirmed that the directly purified protein contains both forms of YqcA that are in slow exchange with each other. These results demonstrate that YqcA binds FMN with high affinity which is similar to other flavodoxins [15], [17].…”

“…These results, in combination with the bioinformatics analysis, strongly support that YqcA is a member of the short-chain flavodoxin subfamily. As a part of our systematic investigations on E. coli flavodoxins, we have previously reported the solution structures and backbone dynamics of another E. coli short-chain flavodoxin MioC by NMR spectroscopy [17]. A comparison of the structures between YqcA and MioC shown in Figure 7A–B.…”

“…Up to date, biochemical and structural studies have been performed on these two proteins to address the molecular mechanism underlying their cofactor binding and electron transfer processes [4], [5], [14]–[18]. The structures of both apo- and holo-forms of MioC were solved and the backbone dynamics were investigated by solution NMR spectroscopy [17]. The structure of E. coli FldA in its holo-form has been solved by X-ray crystallography and also subjected to hydrogen-deuterium exchange studies by NMR [15], [18], whereas the apo-form was unable to get crystalized and remain less well characterized.…”

Biochemical characterization of ferredoxin-NADP+reductase interaction with flavodoxin in Pseudomonas putida

“…P. putida has just one Fld-encoding gene, whose homolog is annotated as mioC in E. coli, and this gene encodes an electron carrier for biotin synthesis (8). The role of the Fld (mioC) in P. putida has not been determined (3,9). Due to the number of different physiological roles for both Fd and Fld, electron exchange between Fpr and either Fd or Fld can be a very important cellular process in prokaryotic cells.…”