Active site structure of the DOX-domain and determinants of dioxygenase-activity

-ACTIVITY

The N-terminal domain (amino acid 1-620) of PpoA was assigned by sequence homology as member of the myeloperoxidase enzyme family (Daiyasu and Toh, 2000) and by multiple sequence alignments this study could identify an eighteen amino acid long sequence motif (Section 4.3.1.1) within the DOX-domain that is highly specific for all PpoA-homologue enzymes and thus might guide a way for enzyme classification within the myeloperoxidase family. Based on structures of different myeloperoxidases, the N-terminal domain of PpoA was modeled and found to resemble the crystal structures of the characterized heme-dioxygenases PGHS-1 and PGHS-2 (Section 4.3.1.1; Figure 15). The cofactor and the fatty acid substrate (linoleic acid) were placed in the same position as it is found in PGHS, which leads to a spatial arrangement, in which all known determinants of reactivity are placed in reasonable geometry. The heme is coordinated by a proximal histidine (His377) and on the distal side a second histidine (His202) can be found. The corresponding positions of both histidines were already shown to be crucially involved in enzyme activity of the homologue 7,8-LDS (Garscha and Ernst H Oliw, 2008) and PpoA’s His377Ala-variant is also inactive (Dr. Florian Brodhun, personal communication). The fatty acid is bound within a hydrophobic channel (Figure 17 A) of which Val328 next to the fatty acids C4 has been implemented to be involved in the regioselectivity of hydroperoxide formation (Garscha and Ernst H Oliw, 2009; Brodhun et al., 2010). Besides hydrophobic interactions of the fatty acid tail with apolar amino acids lining the substrate channel, the carboxylate of the substrate might be bound by ionic interactions to the side chain of a basic amino acid. A similar binding mechanism is proposed for both homologue heme dioxygenase: α-DOX and PGHS (Koszelak-Rosenblum et al., 2008). Although at the position homologue to PGHS-2’s Arg120 no basic amino acid was identified within the predicted PpoA-structure, the side chain of Arg336 is

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located at the entrance of the proposed substrate channel and resides at the same distance to the substrate’s carboxylate as Arg120 in mPGHS2 (Figures 15 C and 17 A). Therefore, it was assumed that this residue might confer affinity of the fatty acid substrate to PpoA’s DOX-domain. To validate this hypothesis, a respective variant with an uncharged amino acid at position 336 (Arg336Met) was constructed and its substrate affinity was measured by a kinetic approach and compared to wild type enzyme (Section 4.4.1, Figure 18). Although the measured kinetics of the variant are altered in a way supporting the hypothesis of Arg336 being involved in substrate binding, the effects of this mutation and especially the increase of km are not as pronounced as one would expect for a residue playing a pivotal role in substrate binding. On the one hand, the small magnitude of the observed effect might be either caused by the enzyme’s complex kinetics, which is governed by activation- and inactivation-processes that prevent reaching steady state conditions and hence complicate the evaluation of the kinetics by Michaelis-Menten theory (discussed in more detail in section 5.2), or by the fatty acid substrate forming micelles leading to an underestimation of the true km. Besides these putative systematic errors, one should also keep in mind that the shape of the dioxygen depletion kinetics measured for the Arg336Met-variant indicates a transition towards a different oxidation-mechanism caused by this mutation. Although a sigmoidal description of the measured data leads to a significantly improved fit, a clear conclusion why the conducted mutation should affect the enzyme’s cooperativity in such a drastic way is not easily derived. Interestingly, sigmoidal description of the measured variant-kinetics does not lead to changed values of km and vmax, relative to the wild type values, but affects merely the Hill coefficient. While this effect might be explainable for the Arg336Met-variant, a variant proposed to have affected binding properties;

the observation that the Tyr327Phe-variant possesses a similar effect (Figure 19) renders this hypothesis implausible again and suggests that a lower activity may just unmask an intrinsic cooperative enzyme behavior. Albeit this possibly changed enzyme cooperativity, the km and vmax

of the Arg336Met-variant, as compared to the wild type, are also not altered more significantly for the sigmoidal described kinetics. Thus the small observed effect could have a mechanistic implication and point out that the basic amino acid in the DOX-domain of PpoA rather resembles the function of Arg120 in PGHS-2 than that of the homologue position in PGHS-1. In isoform 2 of this heme DOX the positively charged side chain of Arg120 does not ionically interact with the substrate, but stabilizes its position by formation of weaker hydrogen bonds and thus renders hydrophobic interactions with the fatty acid tail relatively more important (Rieke et al., 1999). In line with other determinants crucially involved in positioning of the fatty acid are oxidations of unusual substrates (e.g. 14:1^Δ9Z, 16:1^Δ9Z, 18:1^Δ8Z, 18:1^Δ11Z, 18:1^Δ12Z and 20:1^Δ11Z), which indicated that the regiospecificity of the oxidation might be determined rather by the distance from the double bond (system) than by the distance from the substrate’s carboxyl- or ω-end (Brodhun et al., 2009; Ernst H Oliw et al., 2011). This finding implicates that the regioselectivity of the dioxygenation within the DOX-domain might depend on the correct placement of the substrate’s double bonds. Based on this observation, Tyr327 can be identified as solely aromatic residue putatively determining the placement of the substrate by -stacking to assure that the

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dioxygenation occurs regio-selective. Remarkably, this residue is highly conserved throughout all dioxygenases and was suggested to be involved in a hydrogen bonding network that places the catalytic competent tyrosyl radical in a position that resembles a rotamer perfectly placed to abstract hydrogen from the fatty acid substrate (Rogge et al., 2006). In contrast to this clear attribution of a certain role, Thuresson et al. speculated that the corresponding COX-residue might rather be involved in placement of the substrate than positioning of the oxidizing tyrosyl (Thuresson et al., 2001). This deduction was based on and is strengthened by the observation that in COX and 7,8-LDS only variants in which the Tyr was replaced by Phe, as another aromatic amino acid, remained active (Thuresson et al., 2001; Garscha and Ernst H Oliw, 2008).

Nevertheless, since a narrower ß-proton splitting of the tyrosyl radical was observed in the EPR-spectrum of the Tyr327Phe-variant, which indicates a conformational perturbation of the oxidizing tyrosine, it is reasonable to conclude that Tyr327 in PpoA contributes, at least partially, to proper positioning of the oxidizing tyrosyl radical. Hence, a role of this residue in substrate positioning would be not more than an additional one and evaluation of the mechanistic implications of this residue is thus complicated. Moreover, preliminary results characterizing PpoA’s Tyr327Leu-variant, which should not contribute to substrate binding by -stacking, indicated that the regioselectivity of dioxygenation by this variant is not less specific and thus Tyr327 is most likely not crucially involved in substrate-placement. Besides the already mentioned function of Tyr327 to place the oxidizing tyrosine radical in a catalytic competent conformation, the spatial arrangement of Tyr327 next to the substrate’s C8 furthermore suggested that the catalytic tyrosyl radical might be (alternatively) formed at this position and the previously implicated Tyr374 (Brodhun et al., 2009; Fielding et al., 2011) might be only a transient link in a radical chain for intra-molecular electron transfer from Tyr327 to heme compound I. To test this hypothesis, radical distances between the tyrosyl-radicals in the distinct domains were measured by DEER (Section 4.6.2) and compared to the tyrosine distances derived from PpoA’s low resolution quaternary structure (Section 4.6.1). Although the distance distribution obtained for the wild type enzyme indicated the presence of an additional minor distance that could be interpreted as a second or alternative radical site at Tyr327, the distances extracted from the dipolar evolutions measured for the Tyr327Phe-variant were basically identical to the wild type pattern and thus a second radical at this position is rather unlikely (Figure 33). Integrating the results from the DEER-experiment of the Tyr327Phe-variant and the modeled active site structure with solely two tyrosines being in reasonable distance to eventually oxidize the substrate, the assignment of Tyr374 as catalytic competent residue is further strengthened.

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5.1.2ACTIVE SITE STRUCTURE OF THE P450-DOMAIN AND DETERMINANTS OF HYDRO PEROXY