Taxonomic distribution of SbmA/BacA and BacA-like antimicrobial peptide transporters suggests independent recruitment and convergent evolution in host-microbe interactions.

Antimicrobial peptides (AMPs) are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. Several pathogens and nitrogen-fixing legume symbionts depend on transporters called SbmA (or BacA) or BclA (BacA-like) to survive exposure to AMPs. However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood.

Activation of Primary C-H Bonds in Oxidative Cyclizations of Tambjamines Catalyzed by Rieske Oxygenases TamC and TamC.

Tambjamines are complex bipyrrole-containing natural products that possess promising bioactive properties. Although is known to produce both cyclic tambjamine MYP1 and the linear precursor (YP1), the biosynthetic machinery used to catalyze the site-selective oxidative carbocyclization at the unactivated 1° carbon of YP1 has remained unclear. Here, we demonstrate that a three-component Rieske system consisting of an oxygenase (TamC) and two redox partner proteins is responsible for this unprecedented activity on YP1 and potentially, a non-native substrate (BE-18591).

Targeted Genome Mining Facilitates the Discovery of a Promiscuous, Hyperthermostable Amidase from Thermovenabulum Gondwanense with Notable Nylon-Degrading Capacity.

Plastics are ubiquitous in our ecosystems, and microplastic accumulation in the environment is an emerging global health concern. Since available recycling technologies are not economically competitive with primary plastic production, global use is expected to reach 1231 megatons by 2060, with 493 megatons leeching into the environment each year. To identify new nylon-recycling biotechnologies, targeted genome mining was used to identify thermostable enzymes capable of degrading polyamides.

Intramolecular Cyclization and a Retro-Ene Reaction Enable the Rapid Fragmentation of a Vitamin B-Derived Breslow Intermediate.

In solution, analogues of the Breslow intermediate formed during catalysis by benzoylformate decarboxylase (BFDC) undergo rapid, irreversible fragmentation. The ability of BFDC to prevent this reaction and preserve its cofactor is a striking example of an enzyme 'steering' a reactive intermediate towards a productive pathway. To understand how BFDC suppresses the off-pathway reactivity of this Breslow intermediate, a clear mechanistic understanding of the fragmentation reaction is required.

Genome Mining Leads to the Identification of a Stable and Promiscuous Baeyer-Villiger Monooxygenase from a Thermophilic Microorganism.

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous.

Discovery of a Tambjamine Gene Cluster in Suggests Convergent Evolution in Bipyrrole Natural Product Biosynthesis.

While bacterial natural products are a valuable source of therapeutics, the molecules produced by most biosynthetic gene clusters remain unknown. Tambjamine YP1, produced by , is partially derived from fatty acids siphoned from primary metabolism. A structurally similar tambjamine produced by , BE-18591, had not been linked to a gene cluster.

Sulfite-Catalyzed Nucleophilic Substitution Reactions with Thiamin and Analogous Pyrimidine Donors Proceed an SAE Mechanism.

When treated with SO, thiamin undergoes a substitution reaction to release a thiazole leaving group and the corresponding sulfonate. Although this reaction could proceed via a simple S2-like mechanism, a multistep addition-elimination (SAE) mechanism involving the addition of SO to C6' of the 4-aminopyrimidine of thiamin has also been proposed. Although this reaction has potential utility in the synthesis of substituted pyrimidines and provides a direct analogue to reactions catalyzed by thiaminases, a detailed mechanistic picture of the SO-catalyzed cleavage of thiamin has remained elusive.

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase.

Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step.

Competing Protonation and Halide Elimination as a Probe of the Character of Thiamin-Derived Reactive Intermediates.

Decarboxylation reactions from comparable thiamin diphosphate- and thiamin-derived adducts of -(halomethyl)benzoylformic acids in enzymic and non-enzymic reactions, respectively, reveal critical distinctions in otherwise similar Breslow intermediates. The ratio of protonation to chloride elimination from the Breslow intermediate is 10-fold greater in the enzymic process. This is consistent with a lower intrinsic barrier to proton transfer on the enzyme, implicating formation of a localized tetrahedral (sp) carbanion that is formed as CO is produced.

O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer.

Phosphite dehydrogenase (PTDH) catalyzes an unusual phosphoryl transfer reaction in which water displaces a hydride leaving group. Despite extensive effort, it remains unclear whether PTDH catalysis proceeds via an associative or dissociative mechanism. Here, primary H and secondary O kinetic isotope effects (KIEs) were determined and used together with computation to characterize the transition state (TS) catalyzed by a thermostable PTDH (17X-PTDH).

Charge Dispersion and Its Effects on the Reactivity of Thiamin-Derived Breslow Intermediates.

The enzymic decarboxylation of 2-ketoacids proceeds via their C2-thiazolium adducts of thiamin diphosphate (ThDP). Loss of CO from these adducts leads to reactive species that are known as Breslow intermediates. The protein-bound adducts of the 2-ketoacids and ThDP are several orders of magnitude more reactive than the synthetic analogues in solution.

Carbon Kinetic Isotope Effects and the Mechanisms of Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid and CO Incorporation into 1,3-Dimethoxybenzene.

The rate of decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in parallel to the extent that the carboxyl group acquires a second proton (1H). However, the conjugate acid would resist C-C bond breaking as that would lead to formation of doubly protonated CO. An alternative via formation of a higher-energy protonated phenyl tautomer (2H) prior to C-C bond breaking would produce protonated CO, an energetically inaccessible species that can be avoided by transfer of the carboxyl proton to water in the same step.

The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation.

It has previously been established that the deprotonated amino substituent of the pyrimidine of thiamin diphosphate (ThDP) acts as an internal base to accept the C2H of the thiazolium in ThDP-dependent enzymes. The amino group has also been implicated in assisting the departure of the aldehydic product formed after loss of CO from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed.

How Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid Avoids Formation of Protonated CO2.

The decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in acidic solutions. The rate of reaction depends upon solution acidity in a manner that is consistent with the formation of the conjugate acid of 1 (RCO2H2(+)), with its higher energy ring-protonated tautomer allowing the requisite C-C bond cleavage. However, this would produce the conjugate acid of CO2, a species that would be too energetic to form.

Lithium-stabilized nucleophilic addition of thiamin to a ketone provides an efficient route to mandelylthiamin, a critical pre-decarboxylation intermediate.

Mandelylthiamin (MTh) is an accurate model of the covalent intermediate derived from the condensation of thiamin diphosphate and benzoylformate in benzoylformate decarboxylase. The properties and catalytic susceptibilities of mandelylthiamin are the subjects of considerable interest. However, the existing synthesis gives only trace amounts of the precursor to MTh as it is conducted under reversible conditions.

Decarboxylation without CO2: why bicarbonate forms directly as trichloroacetate is converted to chloroform.

Patterns in the observed catalysis of decarboxylation reactions required us to conclude that these reactions involve initial hydration of the carboxylate and subsequent loss of bicarbonate. This raises the important and general question of why CO2 is not formed directly. Reaction profiles for the direct decarboxylation of trichloroacetate were generated with DFT calculations and show no significant barrier to the recombination of the incipient trichloromethide and CO2.

Base-catalyzed decarboxylation of mandelylthiamin: direct formation of bicarbonate as an alternative to formation of CO2.

The decarboxylation of mandelylthiamin is subject to general base catalysis (β = 0.26), an outcome that is inconsistent with the expected dissociative transition state in which CO(2) forms along with a residual carbanion. The results implicate a previously unrecognized associative route in which addition of water to a carboxylate followed by base-catalyzed proton transfer and C-C cleavage produces bicarbonate directly.

Origins of steric effects in general-base-catalyzed enolization: solvation and electrostatic attraction.

Brønsted plots for general-base-catalyzed enolization of aldehydes and ketones show significant negative deviations for the rates of proton removal by sterically hindered amine bases. The origins of the deviations are not apparent from considerations of interactions at the site of the proton transfer. Contrasting behavior is observed in general-base-catalyzed proton removal from an iminium derivative, N1'-methyl-2-(1-hydroxybenzyl)thiamin (NMHBnT), which shows no deviations from the Brønsted correlation for sterically hindered amine bases.