Gunhild E. Siboska

Kinetin — 45 years on

Abstract Kinetin (N 6 -furfuryladenine) was the first cytokinin to be isolated almost 45 years ago from DNA as an artifactual rearrangement product of the autoclaving process. Since then its chemical structure and properties have been well described. Most importantly, a wide variety of biological effects of kinetin, including those on gene expression, on inhibition of auxin action, on stimulation of calcium flux, on cell cycle, and as an anti-stress and anti-ageing compound have been reported. Recently, views on this very well known plant growth factor have undergone substantial modifications. New data have appeared which show that kinetin is formed in cellular DNA as the product of the oxidative, secondary modification of DNA. Although the biological significance of the endogenous kinetin and the molecular mechanisms of its action are not completely understood at present, most of the experimental data point toward kinetin acting as a strong antioxidant in vitro and in vivo, with potential beneficial uses in agriculture and human healthcare.

Evidence for the presence of kinetin in DNA and cell extracts

In contrast to the current view that kinetin (N 6‐furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N 6‐furfuryladenine has electrochemical properties that can be applied for monitoring this modified based by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass‐spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N 6‐furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.

A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA

Recently, we have reported the presence of kinetin (N6‐furfuryladenine) in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We have also found that kinetin has electrochemical properties which can be used for monitoring the level of this modified base in DNA. Here, for the first time, we propose a mechanism for the formation of kinetin in DNA in vivo, based on the analyses of its mass spectra. Since hydroxy radical oxidation at the carbon 5′ of the deoxyribose residue yields furfural, we propose that this aldehyde reacts with the amino group of adenine and, after intramolecular rearrangement, kinetin is formed in vivo. Thus kinetin is the first stable secondary DNA damage product known to date with very well defined cytokinin and anti‐aging properties, linked to oxidative processes in the cell. These results also indicate that N6‐furfuryladenine or kinetin is an important component of a new salvage pathway of hydroxy radicals constituting a ‘free radical sink’. In this way, the cells can neutralize the harmful properties of hydroxyl radical reaction products, such as furfural, and respond to oxidative stress by inducing defence mechanisms of maintenance and repair.

Some unusual nucleic acid bases are products of hydroxyl radical oxidation of DNA and RNA.

There are over 100 modified bases and their derivatives found in RNA and DNA. For some of them, data concerning their properties, synthesis and roles in cellular metabolism are available, but for others the knowledge of their functions and biosynthetic pathways is rather limited. We have analysed the chemical structure of modified nucleosides of DNA and RNA considering mainly their putative synthetic routes. On this basis we suggest, that in addition to enzymatic biosynthetic pathways well established for some odd bases, many rare nucleosides can be recognised as products of random chemical reactions. We identify them as primary or secondary products of the reaction of nucleic acids with hydroxyl radicals, the most active oxidising agent in the cell.

Occurrence, biosynthesis and properties of kinetin (N6-furfuryladenine)

In this paper we review the data on the structure and properties of N6-furfuryladenine (kinetin, K) accumulated during the last forty years. In 1955, kinetin was isolated from DNA as an artifactual rearrangement product of the autoclaving process. Subsequently, its cytokinin activity has been established, demonstrating a wide variety of biological effects, including those on gene expression, inhibition of auxin action, stimulation of calcium flux, the cell cycle, and as an anti-stress and anti-ageing compound. Recently, our views on this very well known plant hormone have changed. There are new data, which show that it occurs in cellular DNA as the product of oxidative, secondary modification and a secondary reaction of DNA. Also new results on the biological function of kinetin have been reported. Various biological effects produced by this hormone in vitro and in vivo have made kinetin even more scientifically interesting and commercially attractive as an ingredient of many beauty cosmetics.

Isolation, crystallization and X-ray analysis of the quaternary complex of Phe-tRNAPhe, EF-Tu, a GTP analog and kirromycin

Kirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF‐Tu). Complexes of the antibiotic, Phe‐tRNAPhe, the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF‐Tu were isolated. Crystallization was achieved at 4°C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 Å, and angles of 54.0°. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe‐tRNAPhe, GDPNP, and Thermus aquaticus EF‐Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464–1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 Å. In both cases, the crystals contain one complex per asymmetric unit.

Cytokinin formation by oxidative metabolism

Summary A new hypothesis for the synthesis of cytokinins in plants is presented. It contradicts the idea that they are synthesised exclusively by microbial symbionts. The paper describes how one cytokinin, kinetin, is associated with plant DNA and appears to be synthesised as a secondary product of DNA oxidation. There is much data in the literature indicating that oxidative metabolism may be the source of many cytokinins in plants.

A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl-tRNAMetf by partial digestion with cobra venom ribonuclease

The translation initiation factor IF-2 of prokaryotic organisms functions in the correct formation of the initiation complex fMet-tRNAfMet:mRNA:30 S ribosomal subunit ] 11. Similar to the role of the elongation factor EF-Tu in the elongation step, IF-2 can be regarded as an a~noacyl-tRNA carrier protein which ensures the correct binding of the first amino acid, formylmethionine, in the ribosomal peptidyl transferase centre. However, the binding constant of IF-2 to fMettRNA is too low to allow isolation of the complex. We have studied the interaction of IF-2 with the initiator tRNA by measuring the effect of the protein on the spontaneous hydrolysis of the aminoacyl ester bond [2]. We showed that IF-2 specifically protected the formylated form of the initiator tRNA, ~dicating that IF-2 interacts with the amino acid acceptor region of the initiator tRNA. Furthermore, we showed that this interaction was independent of GTP. To obtain more detailed information about the structural regions of the initiator tRNA involved in the binding to IF-2, we have employed the method of specific protection by IF-2 against ribonuclease digestion of jet-tRNA.

Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNAPhe

Elongation factor Tu (EF‐Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G‐proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF‐TU binds aminoacylated tRNA (aa‐tRNA) forming the ternary complex EF‐TU: GTP: aa‐tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF‐TU: GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF‐TU: GDPNP and yeast Phe‐tRNAphe after its purification by HPLC.

Identification of an unstable 4-hydroxynoneal modification on the 20S proteasome subunit α7 by recombinant antibody technology.

Numerous cellular functions rely on an active proteasome allowing degradation of damaged or misfolded proteins. Therefore changes in the proteasomal activity have important physiological consequences. During oxidative stress the production of free radicals can result in the formation of 4-hydroxynonenal (HNE) following lipid peroxidiation. The HNE moiety is highly reactive and via a nucleophilic attack readily forms covalent links to cysteine, histidine and lysine side chains. However, as the chemical properties of these amino acids differ, so does the kinetics of the reactions. While covalent linkage through Michael addition is well established, reversible and unstable associations have only been indicated in a few cases. In the present study we have identified an unstable HNE adduct on the α7 subunit of the 20S proteasome using phage display of recombinant antibodies. This recombinant antibody fragment recognized HNE modified proteasomes in vitro and showed that this epitope was easily HNE modified, yet unstable, and influenced by experimental procedures. Hence unstable HNE-adducts could be overlooked as a regulatory mechanism of proteasomal activity and a participating factor in the decreased proteasomal activity associated with oxidative stress.