Lincoln G. Scott

Pathway Engineered Enzymatic de novo Purine Nucleotide Synthesis

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.

[2] Preparation of specifically 2H- and 13C-labeled ribonucleotides

Publisher Summary This chapter discusses the enzymatic synthesis of specifically labeled ribonucleotide triphosphates (NTPs) for the in vitro transcription of RNA strategy. Isotopically labeled RNAs are prepared in transcription reactions using labeled NTPs and T7 RNA polymerase. Uniformly labeled NTPs are readily produced by the phosphorylation of nucleotides isolated from bacterial cultures. While some specific isotopic labeling patterns have been produced by bacterial growth on specifically labeled substrates, a general biochemical synthesis of labeled NTPs offers the advantage of a diversity of isotopic labeling patterns that can be created without metabolic scrambling. By coupling the enzymes from the glycolysis and pentose phosphate pathways with those of nucleotide biosynthesis and salvage, isotopically labeled glucose can be converted into NTPs. The strategy for enzymatic synthesis involves the conversion of glucose into 5-phospho-D-ribosyl-α-l-pyrophosphate (PRPP), which is then converted to three of the nucleoside triphosphates—adenosine triphosphate (ATP), guanosine triphosphate (GTP), and uracil triphosphate (UTP). Cytosine triphosphate (CTP) is prepared from UTP in a second enzymatic reaction catalyzed by CTP synthase (PYRG).

Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise function(s) in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8 and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

RNA Structure Determination by NMR

This chapter reviews the methodologies for RNA structure determination by liquid-state nuclear magnetic resonance (NMR). The routine production of milligram quantities of isotopically labeled RNA remains critical to the success of NMR-based structure studies. The standard method for the preparation of isotopically labeled RNA for structural studies in solution is in vitro transcription from DNA oligonucleotide templates using T7 RNA polymerase and unlabeled or isotopically labeled nucleotide triphosphates (NTPs). The purification of the desired RNA can be performed by either denaturing polyacrylamide gel electrophoresis (PAGE) or anion-exchange chromatography. Our basic strategy for studying RNA in solution by NMR is outlined. The topics covered include RNA resonance assignment, restraint collection, and the structure calculation process. Selected examples of NMR spectra are given for a correctly folded 30 nucleotide-containing RNA.

Enzymatic De Novo Pyrimidine Nucleotide Synthesis

The use of stable isotope labeling has revolutionized NMR studies of nucleic acids, and there is a need for methods of incorporation of specific isotope labels to facilitate specific NMR experiments and applications. Enzymatic synthesis offers an efficient and flexible means to synthesize nucleoside triphosphates from a variety of commercially available specifically labeled precursors, permitting isotope labeling of RNAs prepared by in vitro transcription. Here, we recapitulate de novo pyrimidine biosynthesis in vitro, using recombinantly expressed enzymes to perform efficient single-pot syntheses of UTP and CTP that bear a variety of stable isotope labeling patterns. Filtered NMR experiments on (13)C, (15)N, (2)H-labeled HIV-2 TAR RNA demonstrate the utility and value of this approach. This flexible enzymatic synthesis will make implementing detailed and informative RNA stable isotope labeling schemes substantially more cost-effective and efficient, providing advanced tools for the study of structure and dynamics of RNA molecules.

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pKa values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pKa for ionization of 8azaA at position 38 with the apparent pKa for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pKa values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5′-oxygen–phosphorus bond.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.