1y9d

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spinqualitylabelsall models 1y9d, resolution 2.20Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Pyruvate Oxidase variant V265A from Lactobacillus plantarum

Overview

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.

About this Structure

1Y9D is a Single protein structure of sequence from Lactobacillus plantarum with MG, SO4, NA, TPP and FAD as ligands. Active as Pyruvate oxidase, with EC number 1.2.3.3 Full crystallographic information is available from OCA.

Reference

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A., Wille G, Ritter M, Weiss MS, Konig S, Mantele W, Hubner G, Biochemistry. 2005 Apr 5;44(13):5086-94. PMID:15794646

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