1. Catalytic mechanism of leadzyme

2. Lead toxicity through leadzyme

3. References

4. Further reading

Leadzyme is a small ribozyme (catalytic RNA), which catalyzes the cleavage of a specific phosphodiester bond. It was discovered using an in-vitro evolution study where the researchers were selecting for RNAs that specifically cleaved themselves in the presence of lead.^[1][2] However, since then, it has been discovered in several natural systems.^[3][4] Leadzyme was found to be efficient and dynamic ^[5] in the presence of micromolar concentrations of lead ions.^[6] Unlike in other small self-cleaving ribozymes, other divalent metal ions cannot replace Pb²⁺ in the leadzyme.^[7] Due to obligatory requirement for a lead, the ribozyme is called a metalloribozyme.

Leadzyme has been subjected to extensive biochemical and structural characterization.^[8] The minimal secondary structure of leadzyme is surprisingly very simple . It comprises an asymmetric internal loop composed of six nucleotides and a helical region on each side of the internal loop. The cleavage site of leadzyme is located within a four-nucleotide long asymmetric internal loop that also consists of RNA helices on its both sides. This is shown in top figure on right, which is the secondary structure of leadzyme generated using mfold. The structures of leadzyme have also been solved using X-ray crystallography and NMR.^[9][10] The crystal structures of the two conformations of leadzyme are shown in the lower figure on right.

Catalytic mechanism of leadzyme

Leadzyme is thought to perform catalysis using a two-step mechanism.^[11] In the first step of the reaction, the phosphodiester bond is cleaved into two products: 5’ product terminating in 2’3’ cyclic phosphate and the 3’ product in 5’ hydroxyl. This step is similar to other small self-cleaving ribozymes such as the Hammerhead ribozyme and HDV ribozyme.^[12] Both of those ribozymes generate a product, which contain a 2’, 3’ -cyclic phosphate. However, in leadzyme this product is just an intermediate. In the second step of this reaction pathway, the 2’ 3’ -cyclic phosphate undergoes hydrolysis to form 3’ monophosphate. This mode of catalysis is similar to how ribonucleases (proteins) function rather than any known small self-cleaving ribozyme.

The leadzyme is thought to have a highly dynamic structure.^[13][14] Many studies including NMR, X-ray crystallography and molecular modeling have revealed slightly different structures. Recently using time-resolved spectroscopy, it was shown that the active site of leadzyme is very dynamic.^[15] It samples a lot of different conformations in solution and that the delta G of the interconversion between different conformations is very low. Consistent with these studies, a high-resolution crystal structure also revealed two distinct conformations of the leadzyme with different binding sites for Mg²⁺ and Sr²⁺ (Pb²⁺ substitutes) in the two conformations.^[16] In the ground state, leadzyme binds a single Sr²⁺ ion at nucleotides G43, G45 and A45. This binding site is away from the scissile bond (cleavage site) and thus does not explain the involvement of the Pb²⁺ in the catalysis. However, in the second conformation, termed the ‘pre-catalytic’ state, the ribozyme shows two Sr²⁺ binding sites. G43 and G42 interact with one Sr²⁺ whereas the second Sr²⁺ interacts with the A45, C23 and G24. This second Sr²⁺ binding site also potentially interacts with the 2’-OH of the C23 via a water molecule. This second binding site explains how Pb²⁺ could facilitate catalysis by abstracting the 2-OH proton and prepare it for an in-line nucleophillic attack on the scissile phosphate. This is also supported by the fact the reaction of the leadzyme is pH dependent. Thus, Pb²⁺ could be acting as a Lewis acid and activating the 2-OH of C23. The crystal structure is consistent with a two-metal ion mechanism that has been proposed for leadzyme catalysis.^[17]

Lead toxicity through leadzyme

References

Further reading

• {{cite journal | last1 = Pan | first1 = T. | last2 = Uhlenbeck | first2 = O. C. | year = 1992 | title = A small metalloribozyme with a two-step mechanism | url = | journal = Nature | volume = 358 | issue = | pages = 560–563 | doi=10.1038/358560a0 | pmid=1501711}}
• {{cite journal | last1 = Pan | first1 = T. | last2 = Uhlenbeck | first2 = O. C. | year = 1992 | title = In vitro selection of RNAs that undergo autolytic cleavage with lead(2+) | url = | journal = Biochemistry | volume = 31 | issue = | pages = 3887–3895 | doi = 10.1021/bi00131a001 }}
• {{cite journal | last1 = Barciszewska | first1 = M. Z. | last2 = Wyszko | first2 = E. | last3 = Bald | first3 = R. | last4 = Erdmann | first4 = V. A. | last5 = Barciszewski | first5 = J. | year = 2003 | title = 5S rRNA Is a Leadzyme. A Molecular Basis for Lead Toxicity | url = | journal = Journal of Biochemistry | volume = 133 | issue = | pages = 309–315 | doi = 10.1093/jb/mvg042 }}
• {{cite journal | last1 = Kikovska | first1 = E. | last2 = Mikkelsen | first2 = N.-E. | last3 = Kirsebom | first3 = L. A. | year = 2005 | title = The naturally trans-acting ribozyme RNase P RNA has leadzyme properties | journal = Nucleic Acids Research | volume = 33 | issue = | pages = 6920–6930 | doi = 10.1093/nar/gki993 | pmid=16332695 | pmc=1310964}}
• {{cite journal | last1 = Barciszewska | first1 = M. Z. |display-authors=etal | year = 2005 | title = Lead toxicity through the leadzyme | url = | journal = Mutation Research/Reviews in Mutation Research | volume = 589 | issue = | pages = 103–110 | doi = 10.1016/j.mrrev.2004.11.002 }}
• {{cite journal | last1 = Wedekind | first1 = J. E. | last2 = McKay | first2 = D. B. | year = 1999 | title = Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis | doi = 10.1038/6700 | journal = Nature Structural Biology | volume = 6 | issue = | page = 261 }}
• {{cite journal | last1 = Hoogstraten | first1 = C. G. | last2 = Legault | first2 = P. | last3 = Pardi | first3 = A. | year = 1998 | title = NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis | url = | journal = Journal of Molecular Biology | volume = 284 | issue = | pages = 337–350 | doi = 10.1006/jmbi.1998.2182 | pmid=9813122}}
• {{cite journal | last1 = Wedekind | first1 = J. E. | last2 = McKay | first2 = D. B. | year = 2003 | title = Crystal Structure of the Leadzyme at 1.8 Å Resolution:  Metal Ion Binding and the Implications for Catalytic Mechanism and Allo Site Ion Regulation† | url = | journal = Biochemistry | volume = 42 | issue = | pages = 9554–9563 | doi = 10.1021/bi0300783 | pmid=12911297}}
• {{cite journal | last1 = Ferré-D'Amaré | first1 = A. R. | last2 = Scott | first2 = W. G. | year = 2010 | title = Small Self-cleaving Ribozymes | url = | journal = Cold Spring Harbor Perspectives in Biology | volume = 2| issue = | pages = a003574| doi = 10.1101/cshperspect.a003574 | pmc = 2944367}}
• {{cite journal | last1 = Kadakkuzha | first1 = B. M. | last2 = Zhao | first2 = L. | last3 = Xia | first3 = T. | year = 2009 | title = Conformational Distribution and Ultrafast Base Dynamics of Leadzyme | url = | journal = Biochemistry | volume = 48 | issue = | pages = 3807–3809 | doi = 10.1021/bi900256q | pmid=19301929}}
• {{cite journal | last1 = Ohmichi | first1 = T. | last2 = Sugimoto | first2 = N. | year = 1997 | title = Role of Nd3+ and Pb2+ on the RNA Cleavage Reaction by a Small Ribozyme† | url = | journal = Biochemistry | volume = 36 | issue = | pages = 3514–3521 | doi = 10.1021/bi962030d | pmid=9132001}}

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