Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature’s halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA’s diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.

Transfer RNA is one of the most richly modified biological molecules. Biosynthetic pathways that introduce these modifications are underexplored, largely because their absence does not lead to obvious phenotypes under normal growth conditions. Queuosine (Q) is a hypermodified base found in the wobble positions of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using liquid chromatography MS methods, we have screened 1,755 single gene knockouts of Escherichia coli and have identified the key final step in the biosynthesis of Q. The protein is homologous to B 12 -dependent iron-sulfur proteins involved in halorespiration. The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase catalyzes the conversion of oQ to Q in a synthetic substrate, as well as undermodified RNA isolated from an oQ reductase knockout strain. The activity requires inclusion of a reductant and a redox mediator. Finally, exogenously supplied cobalamin stimulates the activity. This work provides the framework for studies of the biosynthesis of other modified RNA components, where lack of accessible phenotype or obvious gene clustering has impeded discovery. Moreover, discovery of the elusive oQ reductase protein completes the biosynthetic pathway of Q.biochemistry | queuosine | reductive dehalogenation N early 100 modifications have been identified in RNA, many of which are found in tRNA and are common to eukaryotes, bacteria, and archaea (1). Most of these modifications are likely not essential under normal laboratory conditions, making discovery of biosynthetic pathways by phenotypic methods impossible. Queuosine (Q), a hypermodified RNA base containing a 7-deazapurine core, is among the more complex RNA modifications described to date. Q replaces the guanine in the wobble positions of the subset of tRNA molecules with a 5′-GUN-3′ sequence in their anticodon loops (His, Asp, Asn, and Tyr). Conservation of Q in RNA of organisms in nearly all kingdoms of life (2) suggests that the modification may be of cardinal importance. A physiological role for Q has eluded discovery partly because of gaps in understanding of the biosynthetic pathway. De novo biosynthesis of Q occurs in bacteria whereas eukaryotes acquire the free base, queuine, from dietary sources (3-5). The bacterial pathway for biosynthesis of Q has been elucidated up to the penultimate intermediate, epoxyqueuosine (oQ) (6, 7). However, the enzyme that catalyzes the final step in the pathway, conversion of oQ to Q, had yet to be identified. Because no selectable phenotypes have been demonstrated for the modification, discovery of the final step in the pathway required a different approach that melded modern analytical methods and emerging tools in bacterial genetics. This methodology has led to successful identification of the final step and can be generalized to other RNA modifications where lack of phenotype has hindered discovery of the biosynthetic pathway.Structural parallels between the 7-deazapurine core of queuosine and 7-deazapurine-containing antibiotic toyocamycin ...