Figure 3.4 Reactions involved in the substrate level ATP production and consumption. (1) succinyl CoA synthetase; (2) hexokinase; (3) phosphofructokinase; (4) phosphoglycerate kinase; (5) pyruvate kinase.
3.5 Biosynthesis of Deoxyribonucleotides
Reduction of the ribose moiety of the ribonucleotide occurs at the ribonucleoside diphosphate level by ribonucleotide reductase (EC 1.17.4.1). The reaction is:
dNDP is phosphorylated to dNTP and used for replication and repair in DNA biosynthesis. It has been shown that ribonucleotide reductase, comprising two large (R1) and two small (R2) subunits, catalyses a rate-limiting step in the production of deoxyribonucleotides required for DNA synthesis. The large subunit (R1) contains the allosteric regulatory sites, while the small subunit (R2) encompasses a binuclear iron centre and a tyrosyl free radical (Elledge et al. 1992). In mammals, defective ribonucleotide reductase often leads to cell cycle arrest, growth retardation, and apoptosis, whereas abnormally increased levels result in higher mutation rates. Similar phenomena have been demonstrated in mutants of A. thaliana. The results suggest that ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and general development in plants (Wang and Liu 2006). The pathway for thymidine nucleotides, which are required for DNA synthesis, is described in Chapter 10
3.6 Nucleic Acid Biosynthesis
DNA polymerases (DNA-directed DNA polymerase, EC 2.7.7.7) synthesize DNA from deoxyribonucleotides. These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase ‘reads’ the existing DNA strand to create two new strands that match the existing one.
These enzymes catalyse the following chemical reaction:
DNA polymerase adds nucleotides to the 3′ end of a DNA strand, one nucleotide at a time. At least 15 classes of DNA polymerase have been identified in animals and terminal deoxyribonucleotidyl transferases. Based on their properties, the polymerases (α to σ) are classified into four families, A, B, X, and Y (Burgers et al. 2001). There are few papers on plant DNA polymerases although there is one which reports that plant DNA polymerase-γ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase (Uchiyama et al. 2004).
Biosynthesis of RNA is catalysed by RNA polymerase (DNA-directed RNA polymerase, EC 2.7.7.6). The reaction is:
RNA polymerase, locally, opens the double-stranded DNA (usually about four turns of the double helix) so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, namely transcription. A transcription factor and its associated transcription mediator complex must be attached to a DNA binding site, a promoter region, before RNA polymerase can initiate the DNA unwinding at that position. RNA polymerase has intrinsic helicase activity, therefore, no additional enzyme is required to unwind the DNA, in contrast to DNA polymerase. RNA polymerase, not only initiates RNA transcription, it also guides the nucleotides into position, facilitates attachment and elongation, has intrinsic proof reading and replacement capabilities, and a termination recognition function. In eukaryotes, RNA polymerase can build chains as long as 2.4 million nucleotides.
Plants have multiple types of nuclear RNA polymerase, each responsible for synthesis of a distinct subset of RNA. All are structurally and mechanistically related to each other and to bacterial RNA polymerase.
RNA polymerase I synthesizes a pre-rRNA 45S (35S in yeast), which matures into 28S, 18S, and 5.8S rRNAs that form the major RNA sections of the ribosome (Grummt 1998). RNA polymerase II synthesizes precursors of mRNAs and most small nuclear RNA (snRNA) and microRNA (miRNA) (Lee et al. 2004). RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol (Willis 1993). RNA polymerase IV synthesizes small interfering RNA (siRNA) in plants (Herr et al. 2005). RNA polymerase V synthesizes RNAs involved in siRNA-directed heterochromatin formation in plants (Wierzbicki et al. 2009).
Eukaryotic chloroplasts contain a plastid-encoded RNA polymerase that structurally is very similar to bacterial RNA polymerase. Chloroplasts also contain a second, structurally unrelated nucleus-encoded RNA polymerase. Mitochondria contain a structurally- and mechanistically-unrelated RNA polymerase that is a member of the single-subunit RNA polymerase protein family.
3.7 Supply of 5-Phosphoribosyl-1-Pyrophosphate
PRPP is an essential substrate for the biosynthesis of nucleotides. In the case of purine nucleotide synthesis, the first step is phosphoribosylamine (PRA) formation from PRPP (Figure 3.1a). In pyridine and pyridine nucleotide biosynthesis, PRPP is used in intermediate steps, namely the formation of nicotinate mononucleotide (NaMN) (step 3 in Figure 3.1b) and OMP (step 4 in Figure 3.1b). In addition to its involvement in de novo nucleotide biosynthesis, PRPP also acts as a substrate in several pathways including salvage biosynthesis of purine, pyrimidine, and pyridine nucleotides as well as tryptophan and histidine biosynthesis (Becker 2001). The enzyme reactions which utilize PRPP as a substrate are summarized in Table 3.2.
PRPP is synthesized from ribose-5-phosphate and ATP by PRPP synthetase (EC 2.7.6.1, ATP: D-ribose-5-phosphate diphosphotransferase). In plants, ribose-5-phosphate is an intermediate in the photosynthetic reductive pentose phosphate cycle (Calvin–Benson–Bassham cycle), as well as the oxidative pentose phosphate pathway. The reductive cycle is the major CO2 fixation pathway. CO2, molecule at a time, is added to the acceptor molecule ribulose-1,5-bisphosphate (RuBP) generating two molecules of 3-phospho-glycerate (Figure 3.5). The oxidative pentose phosphate pathway is an alternative route for oxidizing glucose. As a result, the pentose phosphate pathway is a major source of reducing equivalents for biosynthesis reactions. The pentose phosphate pathway is important for the conversion of hexoses to pentoses such as ribose-5-phosphate (Figure 3.6). Therefore, ribose-5-phosphate is generated both in chloroplasts and the cytosol of plant cells.
Table 3.2 5-Phosphoribosyl-1-pyrophosphate PRPP-utilizing enzymes in plants.
| Enzyme | EC | Reaction |
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Purine nucleotide biosynthesis
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