Paz positiva

The pyrophosphate leaving group in class III appears to be retained after adenylate formation in a pocket which extends beyond the γ phosphate. In class I enzymes pyrophosphate is released after forming the intermediate. This was examined in the adenylation domain of gramicidin S synthetase (Conti et al. 1996). In class II leucyl- or tyrosyl-tRNA synthetases, pyrophosphate is reported to be released after the amino acid adenylate was formed to mediate binding of tRNA which initiates the second step of tRNA amino-alkylation (Lin et al. 1975; Kuratani et al. 2006).

4.3

Conclusion

Adenylate-forming enzymes have important and diverse roles in metabolic pathways of prokaryotes and eukaryotes. We suggest that an inclusive definition of the superfamily based on the commonality of the adenylation chemistry may be more useful than one limited to three dimensional structures. Adenylating enzymes show that the same chemistry can be catalyzed by very different structures. Despite the profound differences in structure and active site architecture, there are clear chemical similarities in the environment that surrounds the α phosphate. This chemical conservation is of course driven by the requirement to stabilize the pentavalent negatively charged transition state. From a chemical standpoint, the nucleophilic attack of the α or γ phosphate by a hydroxyl are closely related, because in both cases a negatively charged nucleophile attacks. For instance, class III enzymes and kinases (as discussed in chapter 2) share a similar coordination of the ATP tri- phosphates, but not the same binding sites for the adenosine moieties, which are located opposite each other. This aligns the α-phosphorous of class III enzymes and the γ-phosphorous of kinases in identical position and hence allows a similar nucleophilic attack at the phosphate.

Chemical conservation has been observed in other enzyme families such as proteases. Though the different families of proteases use different ways to activate a water molecule or another nucleophile to achieve the cleavage of peptide bonds, they all need to stabilize the tetrahedral transition state.

Figure 4.6: Chemical conservation of the serine protease triad. Serine proteases share a catalytic triad (His-Asp- Ser), but are structurally and evolutionary distinct. (a) Trypsin protease (1S81, pork) with catalytic residues H57, D102 and S195. (b) Subtilisin (2ST1, Bacillus amyloliquefaciens) with catalytic residues D32, H64 and S221. (c) Prolyl oligopeptidase (3EQ7, pork) with residues S554, D641 and H680. (d) HCMV protease (1NJU, human herpesvirus 5) with a different catalytic triad: H63, S132 and H157.

Serine proteases, which account for one third of all proteases, for instance comprise the well known catalytic triad (Asp-His-Ser). However serine proteases are structurally diverse as a comparison of trypsin, subtilisin and prolyl oligopeptidase shows (Figure 4.6a-c) (Page and Di Cera 2008). Interestingly, variations of the catalytic triad (e.g. His-Ser-His or Asp-Glu-Ser) are also found (Figure 4.6d) (Polgar 2005). The nucleophilic serine attacks the carbonyl of the substrate (protein or peptide) and forms a covalent acyl-ester intermediate (Hedstrom 2002). The two

assisting residues in the triad are needed to act as a general base (histidine) and an acid (aspartic acid). During reaction the base accepts a proton from the serine hydroxyl moiety and the acid protonates the amino leaving group of the substrate. This arrangement and the oxyanion hole (formed by S221 and N155 in subtilisin) stabilize the formed negative charge of the tetrahedral intermediate (Polgar 2005). Akin to proteases, adenylating enzymes have utilized different spatial arrangements of residues to stabilize the transition state. This is primarily accomplished by the use of one or two positively charged residues such as His, Lys or Arg and the coordination of up to three Mg ions. It is likely that the requirement to activate carboxylic acids for condensation is particularly ancient (thioester, amide and ester bonds are extremely common chemical building blocks) and this may have driven the convergent evolution seen in the structures of the adenylate forming enzymes (Schmelz and Naismith 2009). Furthermore adenylate forming enzymes are not only interesting enzymes, but have utilities in commercial significant biotransformation or biosynthetic chemistry (Watanabe et al. 2006; Challis 2008; Fisch et al. 2009; Meier and Burkart 2009).

4.4

Future work

Class III adenylate-forming enzymes have a three domain topology. Since only two structures (AcsD and AlcC) have been solved, it remains to be proven whether this topology is conserved or not. It therefore is essential to solve more structures of class III enzymes. So far preliminary work was carried out to obtain crystals for AcsA and DesD (see appendix F and G). DesD is another class IIIc and AcsA a class IIIb enzyme. The structure of AcsA would be interesting, since it would be the first structure of a class IIIb enzyme and would reveal whether class IIIb enzymes share a similar topology with class IIIa and IIIc enzymes. Another interesting, yet unanswered, point is the ability of AlcC (as a processive enzyme) to catalyze two or three reactions while AcsD, which shares a homolog topology, just catalyzes one. It is important to take a closer look at the substrate binding sites to identify which residues turns one enzyme into a processive and the other into a single reaction catalyzing one.

Modification of the substrate specificity of other adenylate forming enzymes such as class I enzymes is interesting. For AcsD, a class III enzyme, it was shown that it is capable of utilizing a variety of different types of nucleophiles (-OH, -NH2 and –SH) and that mutation can shift its substrate specificity. The enzymatic synthesized products may have use in biotransformation. The question is whether class I enzymes can be modified to utilize artificial compounds as well. One focus might be on a class I enzyme: the benzoate CoA ligase from Burkholderia xenovorans (Bains

also capable of utilizing fluorinated benzoates but not chlorobenzoates. Modification of its active site might adapt its substrate specificity to allow oxidation of chlorobenzoates as well. Another potential enzyme could be 4-Chlorobenzoate ligase

from Pseudomonas species (Chang et al. 1997). Engineered microorganisms

“equipped” with such enzymes for instance would be valuable tools in bacteriological treatment approaches of chemically polluted areas.