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Phytochelatins (PCs) are known to be the main heavy-metal-detoxifying peptides in the …

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- A papain-like enzyme at work: Native and acyl–enzyme intermediate structures in phytochelatin synthesis

As highlighted by the similarity of their fold and the superimposition of their active site, we have shown that NsPCS belongs to the papain superfamily of cysteine proteases. It is therefore expected that the peptide hydrolase activity of NsPCS, i.e., the deglycination of GSH, resembles the general and well known mechanism of papain-like cysteine proteases. By analogy with such a mechanism, Cys-70, His-183, and Asp-201 (equivalent to Cys-56, His-162, and Asp-180 in AtPCS1) correspond to the catalytic triad in NsPCS. Moreover, it is also highly probable that Cys-70 and His-183 form a thiolate–imidazolium ion pair, stabilized by Asp-201, and that the nucleophilic attack of Cys-70 on the Cys—Gly peptide bond is favored by the oxyanion hole, made up of Cys-70 and Gln-64. In a second step, the water molecule W153, whose nucleophilicity is enhanced by the proximity of His-183 and Asp-201, is then ideally placed to attack the thioester bond and liberate the γ-glutamylcysteine. We could trap the acyl–enzyme complex at very acidic pH (≈3). The pKa values characteristic of the formation of the thiolate–imidazolium ion pair (Cys-S/(His)-ImH+) of papain-like proteins are pKa1 ≈ 3 and pKa2 ≈ 8. pKa1 was found as low as 2.5 in some papains (38), which proves that the formation of the thiolate–imidazolium ion pair and therefore the acylation step can occur even at a pH of ≈3. On the other hand, at this pH, the imidazolium group remains protonated. As a result, His-183 cannot act as a base, which makes the catalytic water molecule W153 unreactive and the acyl–enzyme stable. In a similar way, Wilmouth and colleagues (35, 36) succeeded in stabilizing at pH 5 an acyl–enzyme intermediate between a serine protease, the porcine pancreatic elastase, and the heptapeptide human β-casomorphin-7. We have also determined that the carbonyl oxygen of the thioester linkage is distorted from planarity and that the catalytic water molecule W153 forms an angle close to the expected Bürgi's angle for a nucleophilic attack on a carbonyl bond. The resulting geometry suggests that, as in the serine and cysteine protease mechanism, the hydrolysis reaction in NsPCS occurs through a tetrahedral intermediate. Consequently, because the amino acids of the catalytic triad and oxyanion hole are strictly conserved (Fig. 3), the crystal structures of NsPCS and of its acyl–enzyme conjugate enable us to conclude that the deglycination step in all of the PCS must occur through a very similar mechanism to that of a cysteine protease. The cysteine protease catalytic machinery is independent of the addition of any specific ion, which may explain why the cleavage of glycine from GSH in PCS-like proteins is not activated by Cd2+ or other heavy-metal ions (17). The NsPCS acyl–enzyme structure reveals also that the cysteine sulfur atom of the γ-EC moiety is exposed toward the solvent. It proves that the active site can easily accommodate a GSH-S-conjugate and catalyze its deglycination. The degradation of GSH-S-conjugate is currently proposed to be another function of PCS (18).

NsPCS, in its native form or as a complex with GSH, was found dimeric in both types of crystal. Because the dimerization (i) is observed in two different crystal forms, (ii) results in an extended buried area, and (iii) is mediated mainly by hydrophobic contacts, it is very likely that the dimer is also the active form in solution. Many residues are conserved at the dimeric interface in the PCS family (Fig. 3), suggesting that the active form of all PCS-like proteins is also a dimer. It is noteworthy that Grill et al. (5) found out that PCS activity from Silene cucubalus elutes on gel filtration at a volume corresponding to a molecular mass of 95 kDa. Knowing that the molecular mass of PCS molecules falls around 50 kDa, it fits with a dimer, which is in agreement with our result. In the crystallographic dimer, the two monomers differ at several places. First, the catalytic water molecule W153 is visible in only one of them. Second, the observable conformations of Arg-173 and the number of water molecules in close interaction with this residue are different in the two monomers. Finally, the protruding loop, which is rather close to the active site, does not show the same disorder in both monomers. The two monomers are therefore structurally and mechanistically different, which suggests that some cooperativity could exist between them.

The two NsPCS structures are not sufficient to elucidate the mechanism of PC synthesis, i.e., the reaction of another GSH molecule or of a PCn molecule on the acyl–enzyme. NsPCS, unlike all other PCS-like proteins, can hardly synthesize PCn ≥ 2 (11, 19, 20); furthermore, we did not find in the acyl–enzyme structure any other fixed GSH or closely related molecule. The fact that the C-terminal part is absent in NsPCS is not crucial for the PCS activity because the N-terminal domain of AtPCS1 alone is active (10), which suggests that the acceptor binding site is in the N-terminal domain. Furthermore, if we suppose that the PC synthesis mechanism from the acyl–enzyme is similar to the deglycination step, the amino group of the incoming GSH is expected to take the place of the nucleophilic water molecule W153. As a consequence, the second substrate binding site should be close enough to the first GSH binding site. An empty zone, in connection with the cavity occupied by GSH, is actually observed in the acyl–enzyme structure and could accommodate another GSH molecule (Fig. 4). The incoming GSH molecule could be stabilized by conserved residues such as Arg-173, Asp-201, Gln-64, Lys-206, or Tyr-207, all of them involved in the formation of the cavity. Remarkably, two regions around this zone, the B-loops 1 and 3, are well conserved among all of the members of the PCS family, except NsPCS. Notably, Gln-67 (in the B-loop 1) and Arg-180 (in the B-loop 2) are replaced in the other PCS-like proteins by much less volumic residues, a proline and a glycine, respectively. We are therefore tempted to propose that the NsPCS difficulties to synthesize PCs is related to the nature and the orientation of these two loops, which could hinder the binding of the second substrate. On the other hand, the protruding loop, which is highly disordered in the acyl–enzyme complex and close to the active site, could also play a role, because the NsPCS sequence differs also from the consensus sequence in this region.

The role of the conserved cysteines for the PCS activity is currently debated. Four cysteines (referred to as Cys-90, Cys-91, Cys-109, and Cys-113 in PCS from A. thaliana) are conserved in all PCS-like proteins except NsPCS. It has been thought that these cysteines could participate in the fixation of heavy atom ions known to activate the synthesis of PCs (12, 15). Recently, Vatamaniuk et al. (13) showed by site-directed mutagenesis that none of these four cysteines decreased the ability of AtPCS1 to confer Cd2+ tolerance or mediate PC synthesis. Tsuji et al. (11) confirmed these results by showing that the activity of the AtPSC1 N-terminal domain remains unchanged when the four cysteines are replaced by the NsPCS corresponding residues Ala, Val, Arg, and Ser. We found that these four residues are very close to each other in the NsPCS structure. Based on the sequence alignment of Fig. 3, we can suppose that the corresponding cysteines in eukaryotic PCS form a “cysteine patch” stabilized by disulfide bonds. As illustrated in Fig. 4, this cysteine patch would be far from the active site and also from the putative acceptor binding site. Some care remains required because the region around Ala-111 and Val-112 is strongly unconserved, and a misalignment of the NsPCS sequence is conceivable. Nevertheless, the analysis of the NsPCS structures tends to confirm that these four cysteines residues are not determining for the PC synthesis, as pointed out by Vatamaniuk et al. (13) and Tsuji et al. (11).

In conclusion, we have proved that NsPCS belongs structurally and mechanistically to the papain superfamily of cysteine proteases. A catalytic triad and an oxyanion hole are responsible for the deglycination of a GSH donor molecule with a mechanism similar to that described for cysteine proteases. The attack of the acceptor molecule on the acyl–enzyme intermediate remains uncertain. However, the analysis of the NsPCS structures enables us to propose a location for the acceptor binding site and also to give some structural clue as to why, unlike the other PCS-like proteins, NsPCS synthesizes only very weakly PCs. The orientation of specific loops close to the active site could indeed obstruct the binding of the acceptor GSH molecule. Both structures also bring a new element about the role of conserved cysteine residues. In the NsPCS structure, these cysteines are far from the active sites, which supports the view that they are not crucial for PC synthesis. Finally, we propose that, as in the crystal, NsPCS is dimeric in solution. Some subtle but significant differences between these two dimers also suggest some cooperative mechanism in PCs synthesis.



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