Emily J. Parker

The enzyme MetX is a homoserine O-acetyltransferase that catalyzes the first step in methionine biosynthesis and is essential for survival and virulence of various pathogens. It is an attractive target for antifungal and antibacterial drug development. MetX catalyzes the acetyl transfer from acetyl-CoA (AcCoA) to homoserine via a ping-pong mechanism involving an acyl-enzyme intermediate. The active site contains a Ser-His-Asp catalytic triad, which constitutes its core catalytic machinery. Here we investigated the mechanistic details of MetX from Mycobacterium tuberculosis (MtbMetX) using a combination of quantum mechanics/molecular mechanics (QM/MM) calculations, mutagenesis, and mass spectrometry. QM/MM calculations suggest that D320 of the catalytic triad participates in the proton transfer during homoserine acetylation, but not during acyl-enzyme formation. Experiments showed that a D320N substitution, which removes the proton-accepting capability of D320 as well as the pK_a modulation of H350 by D320, still allowed acyl-enzyme formation at a markedly reduced rate, but significantly impaired the production of acetyl-homoserine. To isolate the effect of D320's participation in proton transfer from its pK_a modulation role, we used QM/MM calculations to simulate a system where D320 could modulate H350 pK_a but not accept a proton. These calculations suggest that while D320's proton-accepting role is not required for the AcCoA reaction, it contributes thermodynamically in the homoserine reaction by lowering the energy of the forward pathway. Elucidating the mechanistic details of MtbMetX reactions offers valuable insights that will facilitate the development of mechanism-based inhibitors, contributing to future therapeutic strategies.

We demonstrate that cluster-encoded but unrelated indole diterpene cyclases from different fungal classes catalyse a tetrahydropyran ring formation critical for efficient indole diterpene biosynthesis.

Terpene cyclases (TCs) catalyze the generation of complex polycyclic terpenoids through elaborate mechanistic cascades. Very little is known about the unusual type II TCs responsible for generation of a large diverse family of meroterpenoids known as indole diterpenoids (IDTs). The radarins are insecticidal and cytotoxic IDTs that possess an unusual core architecture for which the catalytic machinery is unknown. Here, we interrogate the genome of Aspergillus fresenii to reveal the biosynthetic machinery responsible for delivering the specific radarin scaffold. We demonstrate that a bespoke regioselective IDT epoxidase (RadM) acts at the terminal olefin of precursor 3'-geranylgeranylindole, providing access to this distinct class of IDT architectures. Coexpression of this epoxidase with a noncanonical type II TC RadB led to the biosynthesis of radarin C, in a complex skeletal rearrangement that proceeds via rare alcoholic deprotonation and ketone formation. Our modeling of RadB reveals that catalysis by RadB is enabled by a unique His residue that acts as both the proton donor and acceptor. Intriguingly, coexpression of RadM with an alternative A. fresenii IDT cyclase gene, which does not share this catalytic His, revealed an alternate partial cyclization of 14,15-epoxy-3'-GGI, producing a new decalin IDT. This work solves the final piece of the biosynthetic puzzle to access the full suite of known IDT architectures, informs type II cyclase engineering efforts, and expands the repertoire of impressive chemistry catalyzed by noncanonical TCs.

Indole terpenoids make up a large group of secondary metabolites that display an enticing array of bioactivities. While indole diterpene (IDT) and rarely indole sesquiterpene (IST) pathways have been found individually in filamentous fungi, here we show that both cluster types are encoded within the genome of Tolypocladium album. Through heterologous reconstruction, we demonstrate the SES cluster encodes for IST biosynthesis and can tailor IDT substrates produced by the TER cluster.

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from Aspergillus striatus and Aspergillus desertorum. The function of four putative cyclases was interrogated via heterologous expression. Two specific cyclases were identified that catalyze the formation of epimers emindole SA and DA from A. striatus and A. desertorum, respectively. These cyclases are both clustered along with all the elements required for basic IDT biosynthesis yet catalyze an unusual Markovnikov-like cyclization cascade with alternate stereochemical control. Their identification reveals that these alternate architectures are not generated by mechanistically sloppy or promiscuous enzymes, but by cyclases capable of delivering precise regio- and stereospecificities.

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho-d-ribose 1-diphosphate (PRPP) binds to the C-terminal domain and coordinates to Mg2+, in a site completed by two flexible loops. Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20–21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism. This article is part of the themed issue ‘Reactivity and mechanism in chemical and synthetic biology’.

Nodulisporic acids (NAs) are structurally complex potent antiinsectan indole diterpenes. We previously reported the biosynthetic gene cluster for these metabolites in Hypoxylon pulicicidum and functionally characterised the first five steps of the biosynthetic pathway. Here we reveal a highly complex biosynthetic array, furnishing multiple end products through expression of cluster components in Penicillium paxilli. We show that seven additional cluster‐encoded gene products comprise the biosynthetic machinery that elaborate precursor NAF in this highly branched pathway. The combined action of these enzymes delivers 37 NA congeners including four major end products, NAA, NAA₁, NAA₂ and NAA₄. The plethora of intermediates arises due to modification of the carboxylated prenyl tail by a single promiscuous P450 monooxygenase, NodJ, a pivotal branchpoint enzyme which produces four distinct biosynthetic products giving rise to the complex metabolic grid that characterises NA biosynthesis.