The Role of Val-265 for Flavin Adenine Dinucleotide (FAD) Binding in Pyruvate Oxidase:  FTIR, Kinetic, and Crystallographic Studies on the Enzyme Variant V265A^,

Abstract

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

One Step Electrode Fabrication for Direct Electron Transfer Cholesterol Biosensor Based on Composite of Polypyrrole, Green Reduced Graphene Oxide and Cholesterol Oxidase

“…In case of ChOx‐gRGO‐PPy, the fundamental peaks of PPy and gRGO remained unaltered but the new peaks at 1544 cm −1 and 1651 cm −1 could be attributed to amide II linkage or FAD linkage and amide I . This indicated the possibility of covalent bonding of amines of ChOx to acid groups of gRGO . This might be the facilitator for observed DET, along with better stability of ChOx in this biosensor.…”

Heterologous Expression, Purification, and Characterization of an l -Ornithine N 5 -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

“…At 25°C, the NADPH and FAD ϩ equilibrium dissociation constants were 105.6 Ϯ 6.0 M and 9.9 Ϯ 0.3 M, respectively. By contrast, flavoenzymes that contained tightly bound FAD ϩ , such as p-hydroxybenzoate hydroxylase and pyruvate oxidase, have dissociation constants in the nanomolar range for FAD ϩ (13,26). The relatively low affinity of PvdA for FAD ϩ accounts for the lack of characteristic flavin absorbance between 400 and 500 nm in purified recombinant PvdA.…”

Flavin–Protein Complexes: Aromatic Stacking Assisted by a Hydrogen Bond

“…44−46 The apoprotein displaces the equilibrium toward the unstacked form of FAD by anchoring the 5′-ADP moiety throughout interaction with several amino acids of the consensus sequence located in the positively charged groove of the FAD binding site. The use of the ADP moiety as the major anchoring point to the apoprotein has been observed in several flavoenzymes, 47,54,55 and this phenomenon could be a general feature among the FAD binding proteins. Anchoring the ADP likely restricts the conformational sampling of the cofactor and may help the isoalloxazine to translocate into the active site.…”