Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. The most common activities of natural or in vitro-evolved ribozymes are the cleavage or ligation of RNA and DNA and peptide bond formation. Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.
Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis from activated monomers under very specific conditions, such as an RNA polymerase ribozyme. Mutagenesis and selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds. The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.
Attempts have been made to develop ribozymes as therapeutic agents, as enzymes which target defined RNA sequences for cleavage, as biosensors, and for applications in functional genomics and gene discovery.
Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins, were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures. These ribozymes were found in the intron of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman shared the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA." The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell.
It had been a firmly established belief in biology that catalysis was reserved for proteins. However, the idea of RNA catalysis is motivated in part by the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? The concept of "ribonucleic acids as catalysts" circumvents this problem. RNA, in essence, can be both the chicken and the egg.
In the 1980s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for the splicing reaction, he found that the intron could be spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University, was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component.
Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin – or hammerhead – shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme were made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.
Structure and mechanism
Despite having only four choices for each monomer unit (nucleotides), compared to 20 amino acid side chains found in proteins, ribozymes have diverse structures and mechanisms. In many cases they are able to mimic the mechanism used by their protein counterparts. For example, in self cleaving ribozyme RNAs, an in-line SN2 reaction is carried out using the 2’ hydroxyl group as a nucleophile attacking the bridging phosphate and causing 5’ oxygen of the N+1 base to act as a leaving group . In comparison, RNase A, a protein that catalyzes the same reaction, uses a coordinating histidine and lysine to act as a base to attack the phosphate backbone.[clarification needed]
Like many protein enzymes metal binding is also critical to the function of many ribozymes. Often these interactions use both the phosphate backbone and the base of the nucleotide, causing drastic conformational changes. There are two mechanism classes for the cleavage of phosphodiester backbone in the presence of metal. In the first mechanism, the internal 2’- OH group attacks phosphorus center in a SN2 mechanism. Metal ions promote this reaction by first coordinating the phosphate oxygen and later stabling the oxyanion. The second mechanism also follows a SN2 displacement, but the nucleophile comes from water or exogenous hydroxyl groups rather than RNA itself. The smallest ribozyme is UUU, which can promote the cleavage between G and A of the GAAA tetranucleotide via the first mechanism in the presence of Mn2+. The reason why this trinucleotide rather than the complementary tetramer catalyze this reaction may be because the UUU-AAA pairing is the weakest and most flexible trinucleotides among the 64 conformations, which provides the binding site for Mn2+.
Phosphoryl transfer can also be catalyzed without metal ions. For example, pancreatic ribonuclease A and hepatitis delta virus(HDV) ribozymes can catalyze the cleavage of RNA backbone through acid-base catalysis without metal ions. Hairpin ribozyme can also catalyze the self-cleavage of RNA without metal ions but the mechanism is still unclear.
Ribozyme can also catalyze the formation of peptide bond between adjacent amino acid by lowering the activation entropy.
Although ribozymes are quite rare in most cells, their roles are sometimes essential to life. For example, the functional part of the ribosome, the biological machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. In a model system, there is no requirement for divalent cations in a five-nucleotide RNA catalyzing trans-phenylalanation of a four-nucleotide substrate with 3 base pairs complementary with the catalyst, where the catalyst/substrate were devised by truncation of the C3 ribozyme.
The best-studied ribozymes are probably those that cut themselves or other RNAs, as in the original discovery by Cech and Altman. However, ribozymes can be designed to catalyze a range of reactions (see below), many of which may occur in life but have not been discovered in cells.
Ribozymes and the origin of life
RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule rather than dividing these functions between DNA and protein as they are today; this hypothesis is known as the "RNA world hypothesis" of the origin of life. Since nucleotides and RNA and thus ribozymes can arise by inorganic chemicals, they are candidates for the first enzymes, and in fact, the first "replicators", i.e. information-containing macro-molecules that replicate themselves. An example of a self-replicating ribozyme that ligates two substrates to generate an exact copy of itself was described in 2002.
Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificially-produced self-cleaving RNAs that have good enzymatic activity have been produced. Tang and Breaker isolated self-cleaving RNAs by in vitro selection of RNAs originating from random-sequence RNAs. Some of the synthetic ribozymes that were produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme. In 2015, researchers at Northwestern University and the University of Illinois at Chicago have engineered a tethered ribosome that works nearly as well as the authentic cellular component that produces all the proteins and enzymes within the cell. Called Ribosome-T, or Ribo-T, the artificial ribosome was created by Michael Jewett and Alexander Mankin. The techniques used to create artificial ribozymes involve directed evolution. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcribing them with reverse transcriptase into various cDNA and amplified with error-prone PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are covalently linked to the substrate. If a molecule possesses the desired ligase activity, a streptavidin matrix can be used to recover the active molecules.
Lincoln and Joyce developed an RNA enzyme system capable of self replication in about an hour. By utilizing molecular competition (in vitro evolution) of a candidate RNAmixture, a pair of ribozymes emerged, in which each synthesizes the other by joining synthetic oligonucleotides, with no protein present.
Although not true catalysts, the creation of artificial self-cleaving riboswitches, termed aptazymes, has also been an active area of research. Riboswitches are regulatory RNA motifs that change their structure in response to a small molecule ligand to regulate translation. While there are many known natural riboswitches that bind a wide array of metabolites and other small organic molecules, only one ribozyme based on a riboswitch has been described, glmS. Early work in characterizing self-cleaving riboswitches was focused on using theophylline as the ligand. In these studies an RNA hairpin is formed which blocks the ribosome binding site, thus inhibiting translation. In the presence of the ligand, in these cases theophylline, the regulatory RNA region is cleaved off, allowing the ribosome to bind and translate the target gene. Much of this RNA engineering work was based on rational design and previously determined RNA structures rather than directed evolution as in the above examples. More recent work has broadened the ligands used in ribozyme riboswitches to include thymine pyrophosphate (2). Fluorescence-activated_cell_sorting has also been used to engineering aptazymes.
RNA polymerase ribozyme
Modern life, based largely on DNA as the genetic material, is thought to have descended from RNA-based organisms in an earlier RNA world. RNA life would have depended on an RNA-dependent RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Tjhung et al. have obtained an RNA polymerase ribozyme by in vitro evolution that has an unprecedented level of activity in copying complex RNA molecules. However, this ribozyme is unable to copy itself and its RNA products have a high mutation rate. Nevertheless, progress continues to be made towards the goal of obtaining, by in vitro evolution, an accurate, efficient self-reproducing RNA polymerase ribozyme in order to improve understanding of the early evolution of life.
Samanta and Joyce found that a highly evolved RNA polymerase ribozyme was able of function as a reverse transcriptase, that is, it can synthesize a DNA copy using an RNA template. Such an activity is considered to have been crucial for the transition from RNA to DNA genomes during the early history of life on earth. Reverse transcription capability could have arisen as a secondary function of an early RNA dependent RNA polymerase ribozyme.
Ribozymes have been proposed and developed for the treatment of disease through gene therapy (3). One major challenge of using RNA based enzymes as a therapeutic is the short half-life of the catalytic RNA molecules in the body. To combat this, the 2’ position on the ribose is modified to improve RNA stability. One area of ribozyme gene therapy has been the inhibition of RNA-based viruses.
Similarly, ribozymes have been designed to target the hepatitis C virus RNA, SARS coronavirus (SARS-CoV), Adenovirus and influenza A and B virus RNA. The ribozyme is able to cleave the conserved regions of the virus’s genome which has been shown to reduce the virus in mammalian cell culture. Despite these efforts by researchers, these projects have remained in the preclinical stage.
Well validated naturally occurring ribozyme classes:
- GIR1 branching ribozyme
- glmS ribozyme
- Group I self-splicing intron
- Group II self-splicing intron - Spliceosome is likely derived from Group II self-splicing ribozymes.
- Hairpin ribozyme
- Hammerhead ribozyme
- HDV ribozyme
- rRNA - Found in all living cells and links amino acids to form proteins.
- RNase P
- Twister ribozyme
- Twister sister ribozyme
- VS ribozyme
- Pistol ribozyme
- Hatchet ribozyme
Notes and references
- Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR (November 1982). "Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena". Cell. 31 (1): 147–57. doi:10.1016/0092-8674(82)90414-7. PMID 6297745. S2CID 14787080.
- Fedor MJ, Williamson JR (May 2005). "The catalytic diversity of RNAs". Nature Reviews Molecular Cell Biology. 6 (5): 399–412. doi:10.1038/nrm1647. PMID 15956979. S2CID 33304782.
- Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP (May 2001). "RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension". Science. 292 (5520): 1319–25. Bibcode:2001Sci...292.1319J. CiteSeerX 10.1.1.70.5439. doi:10.1126/science.1060786. PMID 11358999. S2CID 14174984.
- Zaher HS, Unrau PJ (July 2007). "Selection of an improved RNA polymerase ribozyme with superior extension and fidelity". RNA. 13 (7): 1017–26. doi:10.1261/rna.548807. PMC 1894930. PMID 17586759.
- Wochner A, Attwater J, Coulson A, Holliger P (April 2011). "Ribozyme-catalyzed transcription of an active ribozyme". Science. 332 (6026): 209–12. Bibcode:2011Sci...332..209W. doi:10.1126/science.1200752. PMID 21474753. S2CID 39990861.
- Hean J, Weinberg MS (2008). "The Hammerhead Ribozyme Revisited: New Biological Insights for the Development of Therapeutic Agents and for Reverse Genomics Applications". In Morris KL (ed.). RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Norfolk, England: Caister Academic Press. ISBN 978-1-904455-25-7.
- Enzyme definition Dictionary.com Accessed 6 April 2007
- Carl Woese, The Genetic Code (New York: Harper and Row, 1967).
- The Nobel Prize in Chemistry 1989 was awarded to Thomas R. Cech and Sidney Altman "for their discovery of catalytic properties of RNA".
- Visser CM (1984). "Evolution of biocatalysis 1. Possible pre-genetic-code RNA catalysts which are their own replicase". Origins of Life. 14 (1–4): 291–300. Bibcode:1984OrLi...14..291V. doi:10.1007/BF00933670. PMID 6205343. S2CID 31409366.
- Pyle AM (August 1993). "Ribozymes: a distinct class of metalloenzymes". Science. 261 (5122): 709–14. Bibcode:1993Sci...261..709P. doi:10.1126/science.7688142. PMID 7688142.
- Freisinger E, Sigel RK (2007). "From nucleotides to ribozymes—A comparison of their metal ion binding properties" (PDF). Coord. Chem. Rev. 251 (13–14): 1834–1851. doi:10.1016/j.ccr.2007.03.008.
- Pyle AM (August 1993). "Ribozymes: a distinct class of metalloenzymes". Science. 261 (5122): 709–14. Bibcode:1993Sci...261..709P. doi:10.1126/science.7688142. JSTOR 2882234. PMID 7688142.
- Lilley DM (October 2011). "Mechanisms of RNA catalysis". Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 366 (1580): 2910–7. doi:10.1098/rstb.2011.0132. JSTOR 23035661. PMC 3158914. PMID 21930582.
- Doudna JA, Cech TR (July 2002). "The chemical repertoire of natural ribozymes". Nature. 418 (6894): 222–8. doi:10.1038/418222a. PMID 12110898. S2CID 4417095.
- Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (August 2000). "The complete atomic structure of the large ribosomal subunit at 2.4 A resolution". Science. 289 (5481): 905–20. Bibcode:2000Sci...289..905B. CiteSeerX 10.1.1.58.2271. doi:10.1126/science.289.5481.905. PMID 10937989.
- Turk RM, Chumachenko NV, Yarus M (March 2010). "Multiple translational products from a five-nucleotide ribozyme". Proceedings of the National Academy of Sciences of the United States of America. 107 (10): 4585–9. Bibcode:2010PNAS..107.4585T. doi:10.1073/pnas.0912895107. PMC 2826339. PMID 20176971.
- Cech, T. R. (2000-08-11). "Structural biology. The ribosome is a ribozyme". Science. 289 (5481): 878–879. doi:10.1126/science.289.5481.878. ISSN 0036-8075. PMID 10960319. S2CID 24172338.
- Altman, Sidney (1990-08-01). "Enzymatic cleavage of RNA by RNA". Bioscience Reports. 10 (4): 317–337. doi:10.1007/BF01117232. ISSN 0144-8463. PMID 1701103. S2CID 12733970.
- Walter, Nils G; Engelke, David R (October 2002). "Ribozymes: Catalytic RNAs that cut things, make things, and do odd and useful jobs". Biologist (London, England). 49 (5): 199–203. ISSN 0006-3347. PMC 3770912. PMID 12391409.
- Supattapone S (June 2004). "Prion protein conversion in vitro". Journal of Molecular Medicine. 82 (6): 348–56. doi:10.1007/s00109-004-0534-3. PMID 15014886. S2CID 24908667.
- Gilbert W (1986). "Origin of life: The RNA world". Nature. 319 (6055): 618. Bibcode:1986Natur.319..618G. doi:10.1038/319618a0. S2CID 8026658.
- Paul, Natasha; Joyce, Gerald F. (2002-10-01). "A self-replicating ligase ribozyme". Proceedings of the National Academy of Sciences. 99 (20): 12733–12740. Bibcode:2002PNAS...9912733P. doi:10.1073/pnas.202471099. ISSN 0027-8424. PMC 130529. PMID 12239349.
- Tang J, Breaker RR (May 2000). "Structural diversity of self-cleaving ribozymes". Proceedings of the National Academy of Sciences of the United States of America. 97 (11): 5784–9. Bibcode:2000PNAS...97.5784T. doi:10.1073/pnas.97.11.5784. PMC 18511. PMID 10823936.
- Engineer and Biologist Design First Artificial Ribosome - Designer ribosome could lead to new drugs and next-generation biomaterials published on July 31, 2015 by Northwestern University
- Lincoln TA, Joyce GF (February 2009). "Self-sustained replication of an RNA enzyme". Science. 323 (5918): 1229–32. Bibcode:2009Sci...323.1229L. doi:10.1126/science.1167856. PMC 2652413. PMID 19131595.
- Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A. & Breaker, R. R. Control of gene expression by a natural metabolite-responsive ribozyme. 4–9
- Lynch SA, Gallivan JP (January 2009). "A flow cytometry-based screen for synthetic riboswitches". Nucleic Acids Research. 37 (1): 184–92. doi:10.1093/nar/gkn924. PMC 2615613. PMID 19033367.
- Tjhung KF, Shokhirev MN, Horning DP, Joyce GF. An RNA polymerase ribozyme that synthesizes its own ancestor. Proc Natl Acad Sci U S A. 2020;117(6):2906-2913. doi:10.1073/pnas.1914282117
- Samanta B, Joyce GF. A reverse transcriptase ribozyme. Elife. 2017;6:e31153. Published 2017 Sep 26. doi:10.7554/eLife.31153
- de Feyter R, Li P (June 2000). "Technology evaluation: HIV ribozyme gene therapy, Gene Shears Pty Ltd". Current Opinion in Molecular Therapeutics. 2 (3): 332–5. PMID 11249628.
- Khan AU (May 2006). "Ribozyme: a clinical tool". Clinica Chimica Acta; International Journal of Clinical Chemistry. 367 (1–2): 20–7. doi:10.1016/j.cca.2005.11.023. PMID 16426595.
- Asha K, Kumar P, Sanicas M, Meseko CA, Khanna M, Kumar B (December 2018). "Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections". Journal of Clinical Medicine. 8 (1): 6. doi:10.3390/jcm8010006. PMC 6351902. PMID 30577479.
- Khanna M, Saxena L, Rajput R, Kumar B, Prasad R (2015). "Gene silencing: a therapeutic approach to combat influenza virus infections". Future Microbiology. 10 (1): 131–40. doi:10.2217/fmb.14.94. PMID 25598342.
- Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC (May 2012). "Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site". Molecular Biotechnology. 51 (1): 27–36. doi:10.1007/s12033-011-9437-z. PMID 21744034. S2CID 45686564.
- Kumar B, Asha K, Khanna M, Ronsard L, Meseko CA, Sanicas M (April 2018). "The emerging influenza virus threat: status and new prospects for its therapy and control". Archives of Virology. 163 (4): 831–844. doi:10.1007/s00705-018-3708-y. PMC 7087104. PMID 29322273.
- Lieber A, He CY, Polyak SJ, Gretch DR, Barr D, Kay MA (December 1996). "Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes". Journal of Virology. 70 (12): 8782–91. doi:10.1128/JVI.70.12.8782-8791.1996. PMC 190975. PMID 8971007.
- Nielsen H, Westhof E, Johansen S (September 2005). "An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme". Science. 309 (5740): 1584–7. Bibcode:2005Sci...309.1584N. doi:10.1126/science.1113645. PMID 16141078. S2CID 37002071.
- Fica SM, Tuttle N, Novak T, Li NS, Lu J, Koodathingal P, Dai Q, Staley JP, Piccirilli JA (November 2013). "RNA catalyses nuclear pre-mRNA splicing". Nature. 503 (7475): 229–34. Bibcode:2013Natur.503..229F. doi:10.1038/nature12734. PMC 4666680. PMID 24196718.
- Sigel A, Sigel H, Sigel RK (2011). Structural and catalytic roles of metal ions in RNA. Metal Ions in Life Sciences. 9. RSC Publishing. pp. vii–ix. doi:10.1039/9781849732512. ISBN 978-1-84973-251-2. PMID 22010266.
- Johnson-Buck AE, McDowell SE, Walter NG (2011). "6. Metal Ions: Supporting Actors in the Playbook of Small Ribozymes". Metal ions: supporting actors in the playbook of small ribozymes. Metal Ions in Life Sciences. 9. pp. 175–96. doi:10.1039/9781849732512-00175. ISBN 978-1-84973-094-5. PMC 3365584. PMID 22010272.
- Donghi D, Schnabl J (2011). "7. Multiple Roles of Metal Ions in Large Ribozymes". Multiple roles of metal ions in large ribozymes. Metal Ions in Life Sciences. 9. pp. 197–234. doi:10.1039/9781849732512-00197. ISBN 978-1-84973-094-5. PMID 22010273.
- Trappl K, Polacek N (2011). The ribosome: a molecular machine powered by RNA. Metal Ions in Life Sciences. 9. pp. 253–75. doi:10.1039/9781849732512-00253. ISBN 978-1-84973-094-5. PMID 22010275.
- Suga H, Futai K, Jin K (2011). "10. Metal Ion Requirements in Artificial Ribozymes that Catalyze Aminoacylation and Redox Reactions". Metal ion requirements in artificial ribozymes that catalyze aminoacylation and redox reactions. Metal Ions in Life Sciences. 9. pp. 277–97. doi:10.1039/9781849732512-00277. ISBN 978-1-84973-094-5. PMID 22010276.
- Wedekind JE (2011). "11. Metal Ion Binding and Function in Natural and Artificial Small RNA Enzymes from a Structural Perspective". Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective. Metal Ions in Life Sciences. 9. pp. 299–345. doi:10.1039/9781849732512-00299. ISBN 978-1-84973-094-5. PMID 22010277.
- Doherty EA, Doudna JA (2001). "Ribozyme structures and mechanisms". Annual Review of Biophysics and Biomolecular Structure. 30: 457–75. doi:10.1146/annurev.biophys.30.1.457. PMID 11441810.
- Joyce GF (2004). "Directed evolution of nucleic acid enzymes". Annual Review of Biochemistry. 73: 791–836. doi:10.1146/annurev.biochem.73.011303.073717. PMID 15189159.
- Ikawa Y, Tsuda K, Matsumura S, Inoue T (September 2004). "De novo synthesis and development of an RNA enzyme". Proceedings of the National Academy of Sciences of the United States of America. 101 (38): 13750–5. Bibcode:2004PNAS..10113750I. doi:10.1073/pnas.0405886101. PMC 518828. PMID 15365187.