Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants.

Cytochrome P450 BM3 monooxygenase is the topic of extensive research as many researchers have evolved this enzyme to generate a variety of products. However, the abundance of information on increasingly diversified variants of P450 BM3 that catalyze a broad array of chemistry is not in a format that enables easy extraction and interpretation. We present a database that categorizes variants by their catalyzed reactions and includes details about substrates to provide reaction context. This database of >1500 P450 BM3 variants is downloadable and machine-readable and includes instructions to maximize ease of gathering information. The database allows rapid identification of commonly reported substitutions, aiding researchers who are unfamiliar with the enzyme in identifying starting points for enzyme engineering. For those actively engaged in engineering P450 BM3, the database, along with this review, provides a powerful and user-friendly platform to understand, predict, and identify the attributes of P450 BM3 variants, encouraging the further engineering of this enzyme.

From a binding module to essential catalytic activity: how nature stumbled on a good thing.

Enzymes are complex macromolecules capable of catalyzing a wide variety of chemical reactions with high efficiency. Nonetheless, biological catalysis can be rudimentary. Here, we describe an enzyme that is built from a simple protein fold. This short protein sequence - almost a peptide - belongs to the ancient SH3 family of binding modules. Surprisingly, this binding module catalyzes the specific reduction of dihydrofolate using NADPH as a reducing cofactor, making this a dihydrofolate reductase. Too small to provide all the required binding and catalytic machinery on its own, it homotetramerizes, thus creating a large, central active site environment. Remarkably, none of the active site residues is essential to the catalytic function. Instead, backbone interactions juxtapose the reducing cofactor proximal to the target imine of the folate substrate, and a specific motion of the substrate promotes formation of the transition state. In this feature article, we describe the features that make this small protein a functional enzyme capable of catalyzing a metabolically essential reaction, highlighting the characteristics that make it a model for the evolution of primitive enzymes from binding modules.

Trimethoprim resistance in surface and wastewater is mediated by contrasting variants of the dfrB gene.

Trimethoprim (TMP) is a low-cost, widely prescribed antibiotic. Its effectiveness is increasingly challenged by the spread of genes coding for TMP-resistant dihydrofolate reductases: dfrA, and the lesser-known, evolutionarily unrelated dfrB. Despite recent reports of novel variants conferring high level TMP resistance (dfrB10 to dfrB21), the prevalence of dfrB is still unknown due to underreporting, heterogeneity of the analyzed genetic material in terms of isolation sources, and limited bioinformatic processing. In this study, we explored a coherent set of shotgun metagenomic sequences to quantitatively estimate the abundance of dfrB gene variants in aquatic environments. Specifically, we scanned sequences originating from influents and effluents of municipal sewage treatment plants as well as river-borne microbiomes. Our analyses reveal an increased prevalence of dfrB1, dfrB2, dfrB3, dfrB4, dfrB5, and dfrB7 in wastewater microbiomes as compared to freshwater. These gene variants were frequently found in genomic neighborship with other resistance genes, transposable elements, and integrons, indicating their mobility. By contrast, the relative abundances of the more recently discovered variants dfrB9, dfrB10, and dfrB13 were significantly higher in freshwater than in wastewater microbiomes. Moreover, their direct neighborship with other resistance genes or markers of mobile genetic elements was significantly less likely. Our findings suggest that natural freshwater communities form a major reservoir of the recently discovered dfrB gene variants. Their proliferation and mobilization in response to the exposure of freshwater communities to selective TMP concentrations may promote the prevalence of high-level TMP resistance and thus limit the future effectiveness of antimicrobial therapies.

Label-Free SERS for Rapid Differentiation of SARS-CoV-2-Induced Serum Metabolic Profiles in Non-Hospitalized Adults.

COVID-19 represents a multi-system infectious disease with broad-spectrum manifestations, including changes in host metabolic processes connected to the disease pathogenesis. Understanding biochemical dysregulation patterns as a consequence of COVID-19 illness promises to be crucial for tracking disease course and clinical outcomes. Surface-enhanced Raman scattering (SERS) has attracted considerable interest in biomedical diagnostics for the sensitive detection of intrinsic profiles of unique fingerprints of serum biomolecules indicative of SARS-CoV-2 infection in a label-free format. Here, we applied label-free SERS and chemometrics for rapid interrogation of temporal metabolic dynamics in longitudinal sera of mildly infected non-hospitalized patients (n = 22), at 4 and 16 weeks post PCR-positive diagnosis, and compared them with negative controls (n = 8). SERS spectral markers revealed distinct metabolic profiles in patient sera that significantly deviated from the healthy metabolic state at the two sampling time intervals. Multivariate and univariate analyses of the spectral data identified abundance dynamics in amino acids, lipids, and protein vibrations as the key spectral features underlying the metabolic differences detected in convalescent samples and perhaps associated with patient recovery progression. A validation study performed using spontaneous Raman spectroscopy yielded spectral data results that corroborated SERS spectral findings and confirmed the detected disease-specific molecular phenotypes in clinical samples. Label-free SERS promises to be a valuable analytical technique for rapid screening of the metabolic phenotype induced by SARS-CoV-2 infection to allow appropriate healthcare intervention.

Epistasis between promoter activity and coding mutations shapes gene evolvability.

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Tuning Selectivity in CalA Lipase: Beyond Tunnel Engineering.

Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications.

Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance.

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

Tag-free, specific conjugation of glycosylated IgG1 antibodies using microbial transglutaminase.

We present an efficient approach for tag-free, site-specific conjugation of a fully glycosylated antibody using microbial transglutaminase (mTG). We created variants of trastuzumab where a single surface-exposed residue of the human crystallizable fragment had been substituted to glutamine, with the objective of enabling site-specific mTG-mediated conjugation with primary amine payloads. MTG reactivity was determined by conjugation to an amino fluorophore, demonstrating effective tag-free conjugation at the newly introduced I253Q site. The conjugation of one payload per antibody heavy chain was confirmed by mass spectrometry. We further demonstrated two-step mTG/click chemistry-based conjugation of I253Q trastuzumab with monomethyl auristatin E. Cytotoxicity and specificity of the resulting antibody-drug conjugate were indistinguishable from trastuzumab conjugated by another method although binding to the neonatal Fc receptor was impaired. The resulting fully glycosylated ADC is unique in that it results from minimal modification of the antibody sequence and offers potential for application to cellular imaging, fluorescence microscopy, western blotting or ELISA.

Integrating dynamics into enzyme engineering.

Enzyme engineering has become a widely adopted practice in research labs and industry. In parallel, the past decades have seen tremendous strides in characterizing the dynamics of proteins, using a growing array of methodologies. Importantly, links have been established between the dynamics of proteins and their function. Characterizing the dynamics of an enzyme prior to, and following, its engineering is beginning to inform on the potential of 'dynamic engineering', i.e. the rational modification of protein dynamics to alter enzyme function. Here we examine the state of knowledge at the intersection of enzyme engineering and protein dynamics, describe current challenges and highlight pioneering work in the nascent area of dynamic engineering.

Assessment of the longitudinal humoral response in non-hospitalized SARS-CoV-2-positive individuals at decentralized sites: Outcomes and concordance.

Introduction: Early in the COVID-19 pandemic, reagent availability was not uniform, and infrastructure had to be urgently adapted to undertake COVID-19 surveillance. Methods: Before the validation of centralized testing, two enzyme-linked immunosorbent assays (ELISA) were established independently at two decentralized sites using different reagents and instrumentation. We compared the results of these assays to assess the longitudinal humoral response of SARS-CoV-2-positive (i.e., PCR-confirmed), non-hospitalized individuals with mild to moderate symptoms, who had contracted SARSCoV-2 prior to the appearance of variants of concern in Québec, Canada. Results: The two assays exhibited a high degree of concordance to identify seropositive individuals, thus validating the robustness of the methods. The results also confirmed that serum immunoglobulins persist ≥ 6 months post-infection among non-hospitalized adults and that the antibodies elicited by infection cross-reacted with the antigens from P.1 (Gamma) and B.1.617.2 (Delta) variants of concern. Discussion: Together, these results demonstrate that immune surveillance assays can be rapidly and reliably established when centralized testing is not available or not yet validated, allowing for robust immune surveillance.

Cross-reactivity of antibodies from non-hospitalized COVID-19 positive individuals against the native, B.1.351, B.1.617.2, and P.1 SARS-CoV-2 spike proteins.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide, with implications on the spread of the pandemic. Characterizing the cross-reactivity of antibodies against these VOCs is necessary to understand the humoral response of non-hospitalized individuals previously infected with SARS-CoV-2, a population that remains understudied. Thirty-two SARS-CoV-2-positive (PCR-confirmed) and non-hospitalized Canadian adults were enrolled 14-21 days post-diagnosis in 2020, before the emergence of the B.1.351 (also known as Beta), B.1.617.2 (Delta) and P.1 (Gamma) VOCs. Sera were collected 4 and 16 weeks post-diagnosis. Antibody levels and pseudo-neutralization of the ectodomain of SARS-CoV-2 spike protein/human ACE-2 receptor interaction were analyzed with native, B.1.351, B.1.617.2 and P.1 variant spike proteins. Despite a lower response observed for the variant spike proteins, we report evidence of a sustained humoral response against native, B.1.351, B.1.617.2 and P.1 variant spike proteins among non-hospitalized Canadian adults. Furthermore, this response inhibited the interaction between the spike proteins from the different VOCs and ACE-2 receptor for ≥ 16 weeks post-diagnosis, except for individuals aged 18-49 years who showed no inhibition of the interaction between B.1.617.1 or B.1.617.2 spike and ACE-2. Interestingly, the affinity (KD) measured between the spike proteins (native, B.1.351, B.1.617.2 and P.1) and antibodies elicited in sera of infected and vaccinated (BNT162b2 and ChAdOx1 nCoV-19) individuals was invariant. Relative to sera from vaccine-naïve (and previously infected) individuals, sera from vaccinated individuals had higher antibody levels (as measured with label-free SPR) and more efficiently inhibited the spike-ACE-2 interactions, even among individuals aged 18-49 years, showing the effectiveness of vaccination.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Methods for enzyme library creation: Which one will you choose?: A guide for novices and experts to introduce genetic diversity.

Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.

The Bacterial Genomic Context of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases Highlights an Emerging Threat to Public Health.

Type B dihydrofolate reductase (dfrb) genes were identified following the introduction of trimethoprim in the 1960s. Although they intrinsically confer resistance to trimethoprim (TMP) that is orders of magnitude greater than through other mechanisms, the distribution and prevalence of these short (237 bp) genes is unknown. Indeed, this knowledge has been hampered by systematic biases in search methodologies. Here, we investigate the genomic context of dfrbs to gain information on their current distribution in bacterial genomes. Upon searching publicly available databases, we identified 61 sequences containing dfrbs within an analyzable genomic context. The majority (70%) of those sequences also harbor virulence genes and 97% of the dfrbs are found near a mobile genetic element, representing a potential risk for antibiotic resistance genes. We further identified and confirmed the TMP-resistant phenotype of two new members of the family, dfrb10 and dfrb11. Dfrbs are found both in Betaproteobacteria and Gammaproteobacteria, a majority (59%) being in Pseudomonas aeruginosa. Previously labelled as strictly plasmid-borne, we found 69% of dfrbs in the chromosome of pathogenic bacteria. Our results demonstrate that the intrinsically TMP-resistant dfrbs are a potential emerging threat to public health and justify closer surveillance of these genes.

Development of sulfahydantoin derivatives as ß-lactamase inhibitors.

Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover.

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

Glutamine-walking: Creating reactive substrates for transglutaminase-mediated protein labeling.

Chemically modified proteins are increasingly being tested and approved as therapeutic products. Batch-to-batch homogeneity is crucial to ensure safety and quality of therapeutic products. Highly selective protein modification may be achieved using enzymatic routes. Microbial transglutaminase (mTG) is a robust, easy to use and well-established enzyme that is used at a very large scale in the food industry such that its efficacy and its safety for human consumption are well established. In the context of therapeutic protein modification, mTG should crosslink one or more glutamines on the target protein with an aminated moiety such as a solubilizer, a tracer or a cytotoxic moiety. mTG has the advantage of being unreactive toward the majority of surface-exposed glutamines on most proteins, reducing sample heterogeneity. The caveat is that there may be no reactive glutamine on the target protein, or else a reactive glutamine may be found in a location where its modification compromises function of the target protein. Here we describe the glutamine-walk (Gln-walk), a straightforward method to create a glutamine-substrate site that is reactive to mTG in a target protein. Iterative substitution of single amino acids to a glutamine is followed by facile identification of reactivity with mTG, where covalent labeling of the target with an aminated fluorophore allows visualization of the most reactive modified targets. The approach is empirical; knowledge of the target protein structure and functional regions facilitates application of the method.