XenA was identified as a NAD(P)H-dependent, intracellular, FMN-containing oxidoreductase that belongs to the OYE family. Based on gel filtration chromatography it was found, that XenA is a homodimeric enzyme with 361 amino acids and a size of 39.8 kDa per monomer.
The crystal structure of the enzyme was refined to 1.5 Å resolution. A single residue (Trp302) was found to deviate from the Ramachandran statistics. This deviation might have functional relevance, as Trp302 is part of the FMN-binding site. The XenA monomer showed the typical (!,")8-barrel fold (see chapter 1.1.3). One monomer is in the asymmetric unit and related to the second monomer by a crystallographic 2-fold axis, so that the barrel openings are facing in approximately opposite directions (see Figure 1.10a). The dimer interface is built by the two !1 helices of the monomers and the two C-termini from helix !F to !H. The Trp358 residue from helix !H of one monomer is part of the active site of the neighboring monomer and vice versa (see Figure 1.10b). In comparison to OYE1 the dimer interface is at the opposite side of the barrel.
The FMN cofactor is bound in a similar way as shown for other OYE members. It is placed at the C-terminal end of the "-barrel with its si-face exposed to the solvent. In Figure 1.11a the interactions between the protein and the cofactor are displayed. His178, His181, Arg231 and Gln99 are in hydrogen bonding distance to N(1), O(2) and N(3) of the isoalloxazine ring. The amide protons of Ala57 and Cys25 as well as the %-sulfhydryl group stabilize O(4) and N(5) of the isoalloxazine ring. Amino acid sequence alignments revealed that XenA shows highest sequence identities to XenA (99.7%), XenD (68.6%) and XenE (44.0%) from Pseudomonas putida KT2440, XenA (97.2%) from Pseudomonas putida IIB, YqjM (39.9%) from Bacillus subtilis, TOYE (38.6%) from Thermoanaerobacter pseudethanolicus and NerA (31.3%) from Agrobacterium radiobacter. These 6 enzymes have a cysteine in hydrogen bonding distance to O(4), whereas all other OYE homologues mentioned in Table 1.1 have a conserved
threonine. Therefore one can assume, that these members form a subgroup of the OYE family. The dimethylbenzene part is stabilized by Met24 on its re-side through hydrophobic interactions and by Trp358 through face-on-edge #-# interactions.
Figure 1.10: Overall structure of XenA. (a) The XenA dimer is displayed as ribbon diagram. In the first monomer the !-helices are displayed in blue and "-sheets are displayed in green. In the bound FMN molecule carbon atoms are light blue, oxygen atoms are red, nitrogen atoms are blue and the phosphorus atom is orange. The second monomer is colored in rainbow with N-terminus in blue and C-terminus in red. The bound FMN is displayed in grey. The figure was generated from the PDB code given in brackets using MacPyMOL. (b) Topology map of XenA: !-helices are displayed as rectangles and "-strands are displayed as arrows. Helices and "-strands are numbered according to their order in the barrel. Letters designate secondary structure elements outside the barrel. The numbers at the beginning and the end of each secondary structure element,
2-cyclohexenone, coumarin and 8-hydroxycoumarin were identified as oxidative substrates, whereas 7-hydroxycoumarin was assumed to be an inhibitor. XenA reduces 2-cyclohexenone ten-fold faster than coumarin and 8-hydroxycoumarin and shows a preference for NADPH over NADH.
Structural studies on ligand binding of the oxidized enzyme were performed with coumarin and 8-hydroxycoumarin (see Figure 1.11b and c).
Figure 1.11: Active site of oxidized XenA. All residues are displayed as sticks. The carbon atoms of side chain and backbone amino acids are in grey, nitrogen atoms in blue, oxygen atoms in red, sulfur atoms in yellow and phosphorus atoms in orange. Hydrogen bonding interactions are displayed as dashed lines in red. The asterisk denotes the tryptophan residue from the adjacent monomer. (a) Active site of oxidized XenA without substrate. The carbon atoms of the FMN are displayed in green. (b) Active site of oxidized XenA in complex with coumarin. The carbon atoms of coumarin are displayed in green and the FMN cofactor is violet. (c) Active site of oxidized XenA in complex with 8-hydroxycoumarin. The carbon atoms of 8-hydroxycoumarin are displayed in green and the FMN cofactor is violet. The figures were generated from PDB codes given in bracket using MacPyMOL. (PDB codes: 2H8X, 2H9O, 2H8Z (Griese et al., 2006))
Coumarin binds nearly coplanar to the isoalloxazine ring and is stabilized by #-# interactions, as it was shown for other OYE family members. The carbonyl oxygen is stabilized by hydrogen bonding interactions with His178 and His181. The !,"-unsaturated double bond is positioned optimally for proton and hydride transfer. The C(4) ("-carbon) of coumarin is positioned directly over N(5) of the FMN whereas the C(3) (!-carbon) is close to Tyr183, which was proposed as a proton donor (see chapter 1.1.5). 8-hydroxycoumarin in contrast is flipped by 180° around the central C(1a)-C(4a) axis in comparison to coumarin. Therefore, the phenolic group is not within hydrogen bonding distance to the histidine pair and the olefinic bond does not lie above the flavin N(5). His181 coordinates the O(1) of the substrate.
This binding situation does not allow hydride transfer from the cofactor to the substrate, as the donor and acceptor atoms are too far apart. It is assumed that 8-hydroxycoumarin is bound as the phenolate ion to the oxidized flavin, because the oxidized flavin can stabilize the phenolate ion much better, than the reduced FMNH2. As the orientation of ligands in the active site is mainly enforced by hydrogen bonding interactions to the histidine pair, the deprotonated substrate may preferentially bind with the phenolate oxygen, while the protonated 8-hydroxycoumarin would bind with the carbonyl oxygen. For this reason a productive binding mode is assumed in the reduced state of the enzyme.
2 Objectives
Xenobiotic Reductase A from Pseudomonas putida 86 is a member of the OYE family of flavoproteins. This class of enzymes shows a broad substrate range and the physiological roles of most of the members are still unclear. The active site residues of the family members reveal considerable variations, which might be responsible for different redox properties and further functional differences. Mechanistic studies of OYE family members can help to better understand catalysis by flavoenzymes and may provide insights helping to develop novel biocatalysts for fine chemicals, pharmaceuticals and environmental biotechnology. The presented work focuses on the reaction mechanism of XenA and its structural basis in comparison to other well-known OYE family members.
The first aim of this work is to characterize the reactivity of XenA-wt by measuring steady-state and single turnover kinetics as well as by determining the reduction potential of XenA-bound FMN. To get a more detailed model for the active site, the crystal structure of oxidized XenA should be improved to achieve true atomic resolution and the structure of reduced XenA should be determined to reveal redox-dependent conformational changes during the catalytic cycle.
The reaction catalyzed by XenA follows a ping-pong mechanism. This implies that the substrates for the reductive and oxidative half-reactions are bound to the same active site and enable a formal hydride transfer between the various compounds and the FMN cofactor. Both substrates are bound in the same position but interact with different active site amino acids.
The main goal of this work is to analyze individual contributions of five different active site residues (Cys25, Tyr27, Tyr183, Trp302 and Trp358) using site-directed mutagenesis, transient kinetics, redox potentiometry and crystal structure analysis.
So far, the structures of substrate complexes of OYE family members are derived from the non-reactive oxidation state of the enzyme. Another aim of this work is to determine the influence of the oxidation state of the FMN on substrate binding and to further elucidate the reaction mechanism, by stabilizing the true Michaelis complexes using site-directed mutagenesis and crystal structure analysis.
3 Synopsis