Leadzyme

File:Secondary Structure of Leadzyme.png

File:The crystal structures of leadzyme.png

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.{{cite journal | last1 = Pan | first1 = T. | last2 = Uhlenbeck | first2 = O. C. | year = 1992 | title = A small metalloribozyme with a two-step mechanism | journal = Nature | volume = 358 | issue = 6387 | pages = 560–563 | doi=10.1038/358560a0 | pmid=1501711| bibcode = 1992Natur.358..560P | s2cid = 4361511 }}{{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+) | journal = Biochemistry | volume = 31 | issue = 16 | pages = 3887–3895 | doi = 10.1021/bi00131a001 | pmid = 1373649 }} However, since then, it has been discovered in several natural systems.{{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 | journal = Journal of Biochemistry | volume = 133 | issue = 3 | pages = 309–315 | doi = 10.1093/jb/mvg042 | pmid = 12761166 }}{{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 = 21 | pages = 6920–6930 | doi = 10.1093/nar/gki993 | pmid=16332695 | pmc=1310964}} Leadzyme was found to be efficient and dynamic {{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 | journal = Biochemistry | volume = 48 | issue = 18 | pages = 3807–3809 | doi = 10.1021/bi900256q | pmid=19301929}} in the presence of micromolar concentrations of lead ions.{{cite journal | last1 = Pan | first1 = T. | last2 = Uhlenbeck | first2 = O. C. | year = 1992 | title = A small metalloribozyme with a two-step mechanism | journal = Nature | volume = 358 | issue = 6387 | pages = 560–563 | doi=10.1038/358560a0 | pmid=1501711| bibcode = 1992Natur.358..560P | s2cid = 4361511 }} Unlike in other small self-cleaving ribozymes, other divalent metal ions cannot replace Pb²⁺ in the leadzyme.{{cite journal | last1 = Arciszewska | first1 = M. Z. |display-authors=etal | year = 2005 | title = Lead toxicity through the leadzyme | journal = Mutation Research/Reviews in Mutation Research | volume = 589 | issue = 2 | pages = 103–110 | doi = 10.1016/j.mrrev.2004.11.002 | pmid = 15795164 }} Due to obligatory requirement for a lead, the ribozyme is called a metalloribozyme.

Leadzyme has been subjected to extensive biochemical and structural characterization.{{cite book|last1=Sigel |first1= Astrid|last2=Operschall|first2=Bert P.|last3=Sigel |first3=Helmut

|chapter= Chapter 11. Complex Formation of Lead(II) with Nucleotides and Their Constituents|pages= 319–402

|publisher= de Gruyter|publication-date= 2017|series= Metal Ions in Life Sciences|volume=17|title=Lead: Its Effects on Environment and Health|year= 2017|editor1-last=Astrid|editor1-first= S.|editor2-last=Helmut|editor2-first=S.|editor3-last=Sigel |editor3-first= R. K. O.|doi=10.1515/9783110434330-011|pmid= 28731304}} 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.{{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 = 3 | pages = 261–268 | pmid = 10074945 | s2cid = 23837 }}{{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† | journal = Biochemistry | volume = 42 | issue = 32 | pages = 9554–9563 | doi = 10.1021/bi0300783 | pmid=12911297}} 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.{{cite journal | last1 = Pan | first1 = T. | last2 = Uhlenbeck | first2 = O. C. | year = 1992 | title = A small metalloribozyme with a two-step mechanism | journal = Nature | volume = 358 | issue = 6387 | pages = 560–563 | doi=10.1038/358560a0 | pmid=1501711| bibcode = 1992Natur.358..560P | s2cid = 4361511 }} 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.{{cite journal | last1 = Ferré-D'Amaré | first1 = A. R. | last2 = Scott | first2 = W. G. | year = 2010 | title = Small Self-cleaving Ribozymes | journal = Cold Spring Harbor Perspectives in Biology | volume = 2| issue = 10 | pages = a003574| doi = 10.1101/cshperspect.a003574 | pmc = 2944367 | pmid=20843979}} 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.{{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 | journal = Journal of Molecular Biology | volume = 284 | issue = 2 | pages = 337–350 | doi = 10.1006/jmbi.1998.2182 | pmid=9813122}}{{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 | journal = Biochemistry | volume = 48 | issue = 18 | pages = 3807–3809 | doi = 10.1021/bi900256q | pmid=19301929}} 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.{{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 | journal = Biochemistry | volume = 48 | issue = 18 | pages = 3807–3809 | doi = 10.1021/bi900256q | pmid=19301929}} 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.{{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† | journal = Biochemistry | volume = 42 | issue = 32 | pages = 9554–9563 | doi = 10.1021/bi0300783 | pmid=12911297}} 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.{{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† | journal = Biochemistry | volume = 36 | issue = 12 | pages = 3514–3521 | doi = 10.1021/bi962030d | pmid=9132001}}

Lead toxicity through leadzyme

Toxic metals like lead are environmental and health hazards and can enter biological systems upon exposure. Lead is a persistent metal and can accumulate in human body over time due to its frequent usage in industries and presence in our environment. Inhalation of lead can have effects that can be range from subtle symptoms to serious illnesses. It is possible that presence of lead in our biological systems can induce catalysis by lead ions.{{cite journal | last1 = Barciszewska | first1 = M. Z. |display-authors=etal | year = 2005 | title = Lead toxicity through the leadzyme | journal = Mutation Research/Reviews in Mutation Research | volume = 589 | issue = 2 | pages = 103–110 | doi = 10.1016/j.mrrev.2004.11.002 | pmid = 15795164 }} Since leadzyme is a relatively simple motif i.e., it has a simple fold, it appears that there are many sequences in the genomes of many natural systems which can potentially fold into a leadzyme structure. A simple search for this RNA motif in the genomes of humans, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana revealed that on average this motif is present with the frequency of 2-9 motifs for 1 Mbp of DNA sequence.{{cite journal | last1 = Barciszewska | first1 = M. Z. |display-authors=etal | year = 2005 | title = Lead toxicity through the leadzyme | doi = 10.1016/j.mrrev.2004.11.002 | journal = Mutation Research/Reviews in Mutation Research | volume = 589 | issue = 2 | pages = 103–110 | pmid = 15795164 }} They also showed that leadzyme motif is very common in the mRNA sequences of these organisms as well. Thus, these sequences could potentially self-cleave in the presence of lead ions. The targeting of these RNA motifs by lead in mRNAs and other RNAs may explain lead-mediated toxicity resulting in cell death.{{cite journal | last1 = Barciszewska | first1 = M. Z. |display-authors=etal | year = 2005 | title = Lead toxicity through the leadzyme | doi = 10.1016/j.mrrev.2004.11.002 | journal = Mutation Research/Reviews in Mutation Research | volume = 589 | issue = 2 | pages = 103–110 | pmid = 15795164 }}

Leadzyme

Catalytic mechanism of leadzyme

Lead toxicity through leadzyme

References

Further reading