João Pedro Marques Sousa

We review state-of-the-art computational modelling of enzyme reaction mechanisms, highlighting hybrid quantum mechanics/molecular mechanics (QM/MM) methods and the emerging role of machine learning in the field.

Morphine and codeine are essential medicines isolated from the opium poppy, whose biosynthesis is limited by the plant's L‐DOPA decarboxylase activity. In the present study, we use molecular dynamics and hybrid quantum mechanics/ molecular mechanics calculations to clarify the L‐DOPA decarboxylase chemical mechanism of decarboxylation and quinonoid intermediate protonation. In addition, the contribution of residues in the periphery of the active site to the rate‐limiting decarboxylation barrier was determined to guide future experimental mutagenesis aimed at increasing the catalytic activity of the aromatic amino acid decarboxylase enzyme, a fundamental step for the opiumpoppy‐free production of BIAs. Our calculations indicate that the decarboxylation barrier is higher in conformations in which the interaction between Tyr350B and the carboxylate leaving group is formed. Furthermore, the barrier for the protonation of the quinonoid intermediate by Tyr350B is predicted to be lower than the decarboxylation and dependent on His205A protonation by the bulk solvent. Residues Glu171, Asp270, Asp313 and Arg482 were identified as possible candidates for rate‐enhancing mutations of P. somniferum aromatic amino acid decarboxylase.

We have studied the hydroxylation mechanism of l-Tyr by the heme-dependent enzyme CYP76AD1 from the sugar beet (Beta vulgaris). This enzyme has a promising biotechnological application in modified yeast strains to produce medicinal alkaloids, an alternative to the traditional opium poppy harvest. A generative machine learning software based on AlphaFold was used to build the structure of CYP76AD1 since there are no structural data for this specific enzyme. After model validation, l-Tyr was docked in the active site of CYP76AD1 to assemble the reactive complex, whose catalytic distances remained stable throughout the 100 ns of MD simulation. Subsequent QM/MM calculations elucidated that l-Tyr hydroxylation occurs in two steps: hydrogen abstraction from l-Tyr by CpdI, forming an l-Tyr radical, and subsequent radical rebound, corresponding to a rate-limiting step of 16.0 kcal·mol–1. Our calculations suggest that the hydrogen abstraction step should occur in the doublet state, while the radical rebound should happen in the quartet state. The clarification of the reaction mechanism of CYP76AD1 provides insights into the rational optimization of the biosynthesis of alkaloids to eliminate the use of opium poppy.

(S)-Norcoclaurine is synthesized in vivo through a metabolic pathway that ends with (S)-norcoclaurine synthase (NCS). The former constitutes the scaffold for the biosynthesis of all benzylisoquinoline alkaloids (BIAs), including many drugs such as the opiates morphine and codeine and the semi-synthetic opioids oxycodone, hydrocodone, and hydromorphone. Unfortunately, the only source of complex BIAs is the opium poppy, leaving the drug supply dependent on poppy crops. Therefore, the bioproduction of (S)-norcoclaurine in heterologous hosts, such as bacteria or yeast, is an intense area of research nowadays. The efficiency of (S)-norcoclaurine biosynthesis is strongly dependent on the catalytic efficiency of NCS. Therefore, we identified vital NCS rate-enhancing mutations through the rational transition-state macrodipole stabilization method at the Quantum Mechanics/Molecular Mechanics (QM/MM) level. The results are a step forward for obtaining NCS variants able to biosynthesize (S)-norcoclaurine on a large scale.

Plant‐derived natural compounds have been used for treating diseases since prehistorical times. The supply of many plant‐derived natural compounds for medicinal purposes, such as thebaine, morphine, and codeine, is primarily dependent on opium poppy crop harvesting. This dependency adds an extra risk factor to ensuring the supply chain because crops are highly susceptible to environmental conditions. Emerging technologies, such as biocatalysis, might help to solve this problem by diversifying the sources of supply of these compounds. Here we review the first committed step in the production of alkaloid painkillers, the production of S‐norcoclaurine, and the enzymes involved. The improvement of these enzymes can be carried out experimentally by directed evolution and rational design strategies, supported by computational methods, to create variants that produce the S‐norcoclaurine precursor for alkaloid painkillers in heterologous organisms, meeting the pharmaceutical industry standards and needs without depending on opium poppy crops.

Fatty acids are crucial molecules for most living beings, very well spread and conserved across species. These molecules play a role in energy storage, cell membrane architecture, and cell signaling, the latter through their derivative metabolites. De novo synthesis of fatty acids is a complex chemical process that can be achieved either by a metabolic pathway built by a sequence of individual enzymes, such as in most bacteria, or by a single, large multi-enzyme, which incorporates all the chemical capabilities of the metabolic pathway, such as in animals and fungi, and in some bacteria. Here we focus on the multi-enzymes, specifically in the animal fatty acid synthase (FAS). We start by providing a historical overview of this vast field of research. We follow by describing the extraordinary architecture of animal FAS, a homodimeric multi-enzyme with seven different active sites per dimer, including a carrier protein that carries the intermediates from one active site to the next. We then delve into this multi-enzyme’s detailed chemistry and critically discuss the current knowledge on the chemical mechanism of each of the steps necessary to synthesize a single fatty acid molecule with atomic detail. In line with this, we discuss the potential and achieved FAS applications in biotechnology, as biosynthetic machines, and compare them with their homologous polyketide synthases, which are also finding wide applications in the same field. Finally, we discuss some open questions on the architecture of FAS, such as their peculiar substrate-shuttling arm, and describe possible reasons for the emergence of large megasynthases during evolution, questions that have fascinated biochemists from long ago but are still far from answered and understood.

The migration of energy production from environmentally hazardous methods to greener methods, exploring renewable sources, is one of the main goals of humanity for the XXI century. Nonetheless, our society is still largely dependent on fossil fuels for the majority of its key activities and will still be for the foreseeable future. This dependence urges the finding of ways to mitigate the environmental impact of the usage of fossil fuels, in particular oil. Sulphur compounds present in crude oil are one of the leading causes of the harmful environmental effects caused by its combustion. For this reason, increasingly lower concentrations of sulphur compounds are demanded, at the governmental level, on the refined oil. Unfortunately, the desulphurization of crude oil, which is currently achieved by chemical hydrodesulphurization, also presents harmful environmental effects. Thus, cleaner methods for desulphurizing crude oil are also needed. Currently, the most promising, with little environmental impact, desulphurization method is the biodesulphurization procedure called the ‘4S pathway’ carried out by three enzymes of the dszABC gene cluster encoded in the plasmid of the bacterium Rhodococcus erythropolis. In addition, a chromosome encoded flavin reductase (DszD) supplies FMNH₂ to the flavin-dependent enzymes of the pathway. Unfortunately, the 4S pathway is not currently attractive for industrial application, due mainly to the low kinetic efficiency of the enzymes that compose it. Therefore, we urgently need to find ways to enhance the 4S pathway, turning it into a viable complement to the current methods of desulphurization of crude oil. In this review, current methods of desulphurization of crude oil are discussed, and the state of the art of the 4S pathway is analyzed. We point out directions and strategies to improve the efficiency of biodesulphurization, to make it attractive and profitable at an industrial scale.

Descoberto um novo vírus, o SARS-CoV-2, em quatro meses foi já possível determinar 182 estruturas de um número muito reduzido de proteínas deste vírus, isoladas ou ligadas a outras entidades, com diferentes técnicas e resoluções. Apresentam-se visualizações adaptadas de algumas proteínas com estruturas já depositadas no Banco de Dados de Proteínas (Protein Data Bank) por cientistas de todo o mundo e através do qual todos colaboram.