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Although AcsD and AlcC have a conserved ATP binding pocket (described in detail in the previous section), their substrate binding site must be different. AcsD, a type A enzyme, uses citric acid and L-serine, but AlcC, a type C enzyme, consumes and performs dimerization of the succinic acid derivatives Sub204 or Sub220. For AcsD, the co-complex of citric acid and the final product citryl-EDA assisted in elucidation of the AcsD reaction mechanism. Unfortunately no such co-complex structures were obtained for AlcC. However, one still can infer substrate binding from the AcsD co- complex structures for a possible ligand location in AlcC. The O1 atom of the citric acid is facing the α-phosphorous in an angle of 151° which is a consistent position for a nucleophile attack to form an enzyme bound citryl-adenylate. Furthermore, two conserved catalytic residues (R305 and H444) are in close proximity to the carboxylate of citric acid. Bearing all these facts in mind the first ternary complex with Sub220 can be modeled in the AlcC ATP co-complex. Sub220 perfectly fits in a small active site pocket and would be coordinated by R312, H162, M447, H449 and D506 (Figure 3.28a). The Sub220 carboxylate and not its amino group would face the α-phosphorous of ATP allowing a nucleophilic attack of the carboxylate. A conserved histidine (H162 in AlcC, H170 in AcsD) is close to coordinate Sub220. In AcsD, H170 is involved in binding of citric acid as well as stacking the adenosine ring of ATP with H444. However in AlcC, H162 is in a less favored position for stacking. The remaining Sub220 binding residues M447 and D506 are only weakly conserved (found in nine other type C enzymes, see also appendix D, Figure 4.5).

Figure 3.28: Docked models of Sub220 and final products in AlcC active site. (a) The docked Sub220 (colored in green) faces with its carboxylate the α phosphate of ATP. Sub220 is coordinated by three conserved residues (H162, R312 and H449) and two weakly conserved residues (M447 and D506). (b) A second Sub220 molecule (colored in purple) faces the Sub220-AMP intermediate (Sub220 moiety colored in green, AMP in yellow) from the solvent accessible face and binds to E473. (c) The Sub220 dimer has to bind in a permutated orientation to allow macrocyclization. (d) The Sub220 dimer is activated by adenylation and a Sub220 dimer intermediate and PPi is generated. (e) A docked alcaligin molecule superposes well with a docked Sub220 dimer (shown in black wireframe).

Similar to AcsD where the L-serine attack must occur on the solvent exposed face of

citric acid, in AlcC only one position for the second substrate molecule is conceivable. In the second ternary complex another Sub220 molecule presumably is coordinated by E473 facing the ester bond of the Sub220-adenylate with its nucleophilic amino group (Figure 3.28b). The Sub220 carboxylate binds to the free amino group of the Sub220-adenylate intermediate. After dimer formation the product has to be released. This would be consistent with mass spectrometry results where a mass peak corresponding to Sub204 dimer is detected although the enzyme was removed by filtration prior analysis. The Sub2042 macrocyclization can happen in two different ways: enzyme catalyzed or autocatalytically. Reactions with Sub204, where AlcC was removed after O/N incubation (to yield sufficient Sub204 dimer product), showed

that the ratio of Sub204 dimer (+ Na+ adduct) to dimer macrocycle peak levels do not

change significantly over a period of 13 days (see appendix E, Figure 4.8). This indicates that AlcC catalyzes the macrocyclization and that the macrocycle is not formed autocatalytically. Therefore a generated dimer would have to bind in a permutated orientation in the active site facing the α-phosphorous of ATP with its free carboxylate (Figure 3.28c). As for the first reaction the dimer has to be activated by adenylation generating an enzyme bound Sub220 dimer intermediate and pyrophosphate (Figure 3.28d). A model of the resulting alcaligin superimposes well

with both substrate position and the macrocycle does not clash with active site residues (Figure 3.28e).

AlcC was shown to form a Sub204 trimer in an open and a closed ring formation. This was quite surprising since the actual biosynthesis model proposes AlcC to form alcaligin, a dimeric macrocycle (Challis 2005; Brickman et al. 2007). A docked model of alcaligin and Sub204 trimer macrocycle shows that the larger macrocycle is still able to fit in the AlcC active site. This is demonstrated by the space filled models of each macrocycle (Figure 3.29a, b). A model of AlcC dimer with two trimeric macrocycles bound reveals that each active site has to work independently since

Figure 3.29: Docked models of alcaligin and the Sub204 trimer in AlcC active site. (a) Space filling model of alcaligin. (b) Space filling model of Sub204 trimer demonstrates that there is still enough room to bind the large trimeric macrocycle. (c) Docked model of two Sub204 trimer macrocycles in both AlcC monomers.

they are too far apart to interact with each other. These models suggest that both reactions (dimerization and macrocyclization) are likely to happen in the same place.

a

b

Figure 3.30: Similarities between AlcC and DesD. (a) Chemical structure of N-hydroxy-N-succinylcadaverine and desferrioxamine E, substrate and product of DesD. (b) A sequence analysis of AlcC and DesD shows that key active site residues (H162, R312, M447, H449, E472, E473, D506 etc.) are conserved in both enzymes.

DesD, another type C enzyme, is involved in biosynthesis of desferrioxamine in Streptomyces coelicolor (Kadi et al. 2007). It has 51 % sequence identity (69 % sequence similarity) with AlcC (see also appendix C, Table 4.5). DesD is proposed to perform trimerization and macrocyclization of N-hydroxy-N-succinylcadaverine to

generate desferrioxamine E (Figure 3.30a). The DesD substrate is a 10- aminocarboxylic compound and differs only by an additional carbon compared to Sub204 (Figure 3.2b), which is a 9-aminocarboxylic substrate. A sequence alignment of both enzymes shows that residues involved in ATP coordination as well as the possible catalytic residues R312 and H449 are found in both enzymes (Figure 3.30b). Residues H162, M447, D506 and E473 which are likely to coordinate Sub220 in AlcC are also present in DesD. Hence the active site must be organized in a similar way. Therefore it is likely that AlcC is able to utilize DesD substrate catalyzing not only dimerization, but also trimerization and macrocyclization.

3.5

Conclusion

The apo and co-complex structure of AlcC are the first structural insights into a NIS family C member. While the adenosine structure was acquired by co-crystallization, the ATP structure was only obtained after replacing the active site bound sulfate. This was accomplished by soaking apo crystals in high concentrations of sodium formate, followed by an ATP/sodium formate solution. The most prominent observation is that ATP is coordinated in a “similar” manner to ATP in AcsD, and that two conserved residues, R312 (that moves in from a remote position) and H449, are found in close proximity to the α-phosphorous. This indicates that these residues, as it was shown for AcsD, are the catalytic residues. Sequence analysis of family C members showed that coordination of the tri-phosphates and the first Mg ion is partly conserved in five binding motifs. The binding site of the second Mg is not conserved and is presumably not found in many other type C enzymes.

AlcC and AcsD share a three domain topology. There are only minor differences in secondary elements such as an added helix (α5) or the extended N-terminal loop in AlcC. Even though they share a similar topology, AlcC has a “head-to-head” dimer and is not able to form the “side-by-side” dimer seen for AcsD due to its shorter L1 loop. The extended N-terminal loop (missing in AcsD) was shown to interlock the two AlcC monomers and stabilize them. It still remains unclear whether dimerization is functionally significant.

their macrocycles. This was unexpected, since preliminary tests in the group of Prof. Challis indicated that with this substrate analogue only a dimer but not a macrocycle was formed. A detailed fragmentation analysis of the MS/MS data illustrated that simple cleavage at amide bonds or elimination of water reproduces the observed fragments. Since no substrate or product co-complex was obtained substrate binding models were created. The docked model of Sub220 substrate perfectly fits in a small cavity facing the α phosphate of ATP with its carboxylate similar to citric acid in AcsD allowing a SN2 like attack to generate a Sub220 adenylate. The model of alcaligin and the trimer macrocycle demonstrate that the active site of AlcC is large enough to accommodate both without any domain movements. This would be consistent with the AcsD structure where no structural movement upon nucleophile binding was

observed. A model of AlcC dimer with two coordinated (Sub204)3 macrocycles

demonstrates that the two active sites of AlcC are too far apart to interact with each other and are not likely to form one “large” active site. Interestingly DesD, another type C enzyme, performs a similar trimerization and macrocyclization and shows a high sequence similarity to AlcC. Key active site residues including the likely substrate coordinating residues are found in both enzymes. DesD is involved in desferrioxamine biosynthesis, a substance also used as a FDA approved drug in medical applications such as iron chelating therapies after repetitive blood transfusions or treatment of chronic iron overload or acute iron intoxications (Hua et al. 2008; Suzuki 2008). But so far no structural data of DesD are published and the acquired structural results on AlcC might help in developing new or modified iron chelating compounds used in medical or commercial applications.

3.6

Future work

AlcC and AcsD have a similar three domain topology, but don’t share the same dimer interface. The dimer interface in AlcC seems at least partly conserved in family C. Site directed mutagenesis of hydrogen bond forming residues involved in stabilizing the dimer and their effect on enzyme activity is required to probe their role. Additional biochemical data such as kinetic parameters or mutational studies on H449 and R312 are desirable.

Structural models of AlcC substrate and product coordination were developed based on structural information of AlcC and AcsD co-complex structures. Hence future work will concentrate on obtaining substrate or product co-complexes. Since soaking has not been successful, a different method or a revised protocol is needed. One possible approach is mutation of likely coordinating residues to enhance substrate binding and/or test substrate analogues that act as active inhibitors. However the main problem seems to be the sulfate containing crystallization condition, which does not allow binding of ATP until sulfate is removed or diluted. So far intensive screening for a sulfate-free condition has failed. Surface reduction entropy (SER) approach has been shown to enhance crystallization of several proteins (Derewenda 2004; Goldschmidt et al. 2007). The surface modified AlcC protein could be re- screened with commercial or customized stochastic screens, where sulfate or phosphate is omitted.

three molecules of N-hydroxy-N-succinylcadaverine to generate desferrioxamine E. Since both substrates are quite similar (just a carbon longer), AlcC might also be able to utilize the DesD substrate. The docked model of the Sub204 trimer shows that there is enough space for larger substrates and products. Hence, finding and testing of other natural or chemically synthesized substrates could increase its use in biotransformation.