Eric Kervio

Structural and functional comparison of HemN to other radical SAM enzymes.

Abstract Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.

Chemical Primer Extension in Seconds

Chemical primer extension (CPE) generates nucleic acid strands elongated by the nucleotides encoded in a template strand in the absence of enzymes. Enzymatic primer extension is a fundamental reaction that underlies both replication and transcription. It is also pivotal for biotechnology, as PCR and dideoxy sequencing are based on primer extension. Furthermore, the formation and template-directed extension of primers is probably a key process in the prebiotic phase of the evolution of life. CPE can produce sequence information with unlabeled or fluorophore-labeled mononucleotides. Compared to polymerase-catalyzed primer extension, CPE is inexpensive as it avoids the cost of enzymes and triphosphate substrates, which makes it attractive for genotyping and sequencing applications. A reduction in cost is critical to meet the

Templating efficiency of naked DNA

1000 genome challenge. The chemical replication or copying reactions underlying CPE have been studied for decades in the context of prebiotic evolution. A drawback of known forms of these reactions is that they are less efficient and much slower than their enzymatically catalyzed counterparts. Polymerase-catalyzed primer extension leads to replication of entire bacterial genomes within minutes, but chemical copying assays are run on the timescale of days to weeks. Attempts have been made to overcome the slow nature of primer generation and CPE steps. Oxyazabenzotriazolides were identified as reactive monomers that reduce the half-life time (t1/2) of CPE involving amino-terminal DNA or RNA by more than an order of magnitude, but all subsequent attempts to accelerate these reactions further appeared to hit a “solid wall” of unreactivity or lead to an unacceptable level of lability among the monomers. Control experiments meant to test the tolerance of the assay for pyridine (whose presence was needed to allow for direct addition of active esters of monomers generated in this solvent) led to an unexpected result. At high millimolar concentrations, pyridine massively accelerates spontaneous replication steps by what appears to be an organocatalytic process. The range of reactions for which “organocatalysis” has been established has increased substantially. Although the phenomenon has been known for a long time, the focus of contemporary research has been on enantioselective reactions catalyzed by substoichiometric amounts of chiral organic molecules. Reactions as different mechanistically as aldol additions and transfer hydrogenation benefit from the presence of organocatalysts. However, transphosphorylation reactions are not among the synthetic transformations frequently catalyzed by organocatalysts. Instead, activation by metal ions, such as the magnesium ions found in the active site of polymerases or minerals, is more common. On the other hand, solid-phase DNA synthesis became rapid (and a successful commercial process) when phosphoramidites were combined with tetrazole as organocatalyst. This catalyst is usually referred to as an “activator”, as it is used above stoichiometric concentration. Our study on CPE employed oxyazabenzotriazolides of deoxynucleotides (1a, 1c, 1g, 1t) and 3’-amino-terminal primers (Scheme 1) to generate phosphoramidate linkages that are isoelectronic to phosphodiesters. Some phosphoramidates are accepted by polymerases. Oxyazabenzotriazolides produce phosphodiesters in RNA-based reactions. ,21] Earlier attempts to employ known catalysts for ligation in this system, including proflavine, had been essentially unsuccessful. For our screens, we transferred the assays involving 1 a–t to magnetic beads (Scheme 2). Excess monomers and buffer

The role and source of 5'-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme.

Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with ≥93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows ≥72% of the correct extension product in all cases, and ≥90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

The strength of the template effect attracting nucleotides to naked DNA

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell‐free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10–20‐fold and showed saturation kinetics with an apparent K m of 25 μM. It is proposed that S‐adenosylmethionine is the source of a 5′‐deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B12‐dependent enzymes. Possible roles of S‐adenosylmethionine as a radical source in higher plants are discussed.

The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA

The transmission of genetic information relies on Watson–Crick base pairing between nucleoside phosphates and template bases in template–primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template–primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd′s range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.

Ribonucleotides and RNA Promote Peptide Chain Growth

The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M−1 h−1. For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying.

Amino Acid‐Specific, Ribonucleotide‐Promoted Peptide Formation in the Absence of Enzymes

All known forms of life use RNA-mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA-dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate-terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.

Effect of Supramolecular Inclusion on the 1,2-Rearrangement of Free Radicals

Nucleic acids and polypeptides are at the heart of life. It is interesting to ask whether the monomers of these biopolymers possess intrinsic reactivity that favors oligomerization in the absence of enzymes. We have recently observed that covalently linked peptido RNA chains form when mixtures of monomers react in salt-rich condensation buffer. Here, we report the results of a screen of the 20 proteinogenic amino acids and four ribonucleotides. None of the amino acids prevent phosphodiester formation, so all of them are compatible with genetic encoding through RNA chain growth. A reactivity landscape was found, in which peptide formation strongly depends on the structure of the amino acid, but less on the nucleobase. For example, proline gives ribonucleotide-bound peptides most readily, tyrosine favors pyrophosphate and phosphodiester formation, and histidine gives phosphorimidazolides as dominant products. When proline and aspartic acid were allowed to compete for incorporation, only proline was found at the N-terminus of peptido chains. The reactivity described here links two fundamental classes of biomolecules through reactions that occur without enzymes, but with amino acid specificity.

Chemical Primer Extension: Efficiently Determining Single Nucleotides in DNA

The free radicals 3-ethoxy-2-(ethoxycarbonyl)-3-oxopropyl (1.) and 3-ethoxy-2-(ethoxycarbonyl)-2-methyl-3-oxopropyl (2.) were generated by photolysis of perester precursors in i) hexane solution, ii) in the presence of β-cyclodextrin, and iii) in NaY zeolite. While free radicals in solution are reluctant to rearrange, they do so when encapsulated in β-cyclodextrin or NaY zeolite. The coenzyme-B12-dependent enzymic rearrangement of methylmalonyl-CoA to succinyl-CoA could be mimicked by photochemical generation of an analogue of the putative intermediate radical in a molecular container.