Enzyme Specificity Research Articles

Functional and structural analyses of IMP-27 metallo-β-lactamase: evolution of IMP-type enzymes to overcome Zn(II) deprivation.

IMP-type metallo-β-lactamases are di-Zn(II) enzymes that can inactivate a wide range of bicyclic β-lactam agents used in clinical practice. IMP-27 shares 82% amino acid sequence identity with IMP-1, the first IMP-type enzyme identified. Herein, we conducted structural determination, kinetic, and chelating agent resistance analyses of IMP-27. Once determined, IMP-27 was then compared to its mutant, namely, G262S, and IMP-1. Crystallographic structural analysis of IMP-27 showed an overall structure comparable to that of IMP-1 and other IMP-type enzymes; the positions of the zinc (Zn) ions varied across enzymes. Kinetic analysis showed that IMP-27 had lower catalytic efficiency against penicillins, ceftazidime, cephalexin, and imipenem than IMP-1; however, it had higher affinity and catalytic efficiency against meropenem, especially in the presence of Zn(II). This suggests that the catalytic site of IMP-27 is optimized to hydrolyze meropenem during molecular evolution at the expense of catalytic efficiency against penicillins. However, Zn(II) content analysis after EDTA treatment revealed no significant difference between enzymes. Moreover, analysis of IMP-27 mutants indicated that the differences in kinetic properties and chelator resistance between IMP-1 and IMP-27 were mainly due to an amino acid substitution at position 262.IMPORTANCEThe residue at position 262 has been reported as a key determinant of substrate specificity in IMP-type enzymes. Among more than 80 IMP-type metallo-β-lactamase (MBL) variants, IMP-27 was the first reported IMP-type MBL isolated from Proteus mirabilis. This enzyme has a glycine residue at position 262, which is occupied by serine in IMP-1. Compared with IMP-1, IMP-27 had a significantly higher affinity and catalytic efficiency against meropenem and improved metal-binding capacity, maintaining its activity under Zn(II)-limited conditions better than IMP-1. The analysis of the IMP-27 mutants indicated that differences between IMP-27 and IMP-1 were mainly due to an amino acid substitution at position 262. In the case of IMP-27, the G262S mutation optimized the catalytic site of IMP-27 for meropenem hydrolysis, at the expense of catalytic efficiency against penicillins.

A novel non-catalytic plasmonic immunoabsorbant assay amplification mechanism

The amplification of signals is dependent upon enzyme/ catalyst. Enzyme activity, specificity, pH condition, enzyme linkers, stability, storage and efficiency are some very important factors that can eventually affect the detection results. We aimed to amplify signals without the assistance of enzyme/ catalyst so that the strategy can be used in the wide range of signal detection including immune-assay. Silver nanocrystals (Ag NCs) grown from self-nucleation are used as chromogens due to their strong plasmon band at ∼ 400 nm. During 140 s of reaction time in presence of 6.10 mM CTAB and 0.61 mM of AgNO3, the 0.57 mM of Ascorbic acid allows Ag over growth on antibody-tagged gold nanospheres (10±0.5 nm) that competes silver atoms from the self-nucleation growth and thus affects the growth kinetics of the latter. By utilizing the opened kinetic window, sensitive detection has been achieved from 6.67 × 10–14 M (1 pg) to 3.33 × 10–9 M (50 ng) using human IgG as a model protein. The non-catalytic plasmonic immunoabsorbant assay we developed herein adds a new possibility of plasmonic NPs for sensitive detection. The detection slope and resolution is higher and better than those via signal amplification through the assistance of enzymes/ catalysts. This work supports the concept that nanoparticles mediated reactions are sensitive enough that they have the potential to go independently without enzymes/ catalyst assistance.

Comprehensive Insights into Microbial Lipases: Unveiling Structural Dynamics, Catalytic Mechanism, and Versatile Applications.

Microbial lipases (MLs) are pivotal biocatalysts in lipid biotechnology due to their diverse enzymatic properties and substrate specificity, garnering significant research attention. This comprehensive review explores the significance of MLs in biocatalysis, providing insights into their structure, catalytic domain, and oxyanion hole. The catalytic mechanism is elucidated, highlighting the molecular processes driving their efficiency. The review delves into ML sources, spanning fungi, yeasts, bacteria, and actinomycetes, followed by a discussion on classification and characterization. Emphasizing the scattered findings in the literature, the paper consolidates the latest information on ML applications across various industries, from food and pharmaceuticals to biofuel production and the paper and pulp industry. The review captures the dynamic landscape of ML research, emphasizing their structure-function relationships and practical implications across diverse sectors.

Large-scale screening identifies enzyme combinations that remove in situ grown oral biofilm

Bacteria in the oral cavity are responsible for the development of dental diseases such as caries and periodontitis, but it is becoming increasingly clear that the oral microbiome also benefits human health. Many oral care products on the market are antimicrobial, killing a large part of the oral microbiome but without removing the disease-causing biofilm. Instead, non-biocidal matrix-degrading enzymes may be used to selectively remove biofilm without harming the overall microbiome.The challenge of using enzymes to degrade biofilms is to match the narrow specificity of enzymes with the large structural diversity of extracellular polymeric substances that hold the biofilm together. In this study, we therefore perform a large-scale screening of single and multi-enzyme formulations to identify combinations of enzymes that most effectively remove dental biofilm.We tested >400 different treatment modalities using 44 different enzymes in combinations with up to six enzymes in each formulation, on in vitro biofilms inoculated with human saliva. Mutanase was the only enzyme capable of removing biofilm on its own. Multi-enzyme formulations removed up to 69 % of the biofilm volume, and the most effective formulations all contained mutanase. We shortlisted 10 enzyme formulations to investigate their efficacy against biofilms formed on glass slabs on dental splints worn by 9 different test subjects. Three of the ten formulations removed more than 50 % of the biofilm volume. If optimal enzyme concentration and exposure time can be reached in vivo, these enzyme combinations have potential to be used in novel non-biocidal oral care products for dental biofilm control.

Discovery of a polyurethane-degrading enzyme from the gut bacterium of plastic-eating mealworms

Although numerous polyurethane (PU)-degrading enzymes were identified from a diverse array of microorganisms in soil or compost, it is intriguing to investigate whether novel PU-degrading enzymes can be discovered in other biological environments. This study reports the discovery of an enzyme (MTL) for PU plastic degradation from the bacterial strain Mixta tenebrionis BIT-26, isolated from the gut of plastic-eating mealworms. MTL shows significant degradation activity towards three commercial PU substrates, including Impranil®DLN-SD, thermoplastic films (PEGA-HDI), and thermoset foams (PEGA-TDI), by cleaving the ester bonds in the polyester polyol moieties. The structure, molecular docking, and site-directed mutagenesis analyses elucidate the substrate binding model. A combination of structure-based comparison and mutational studies reveals the underlying architecture of the enzyme's specificity. These findings provide a fresh perspective into understanding plastic metabolism in the gut of plastic-eating insects and a prospective path for developing a biodegradation technique for plastic waste disposal.

Enzymatic synthesis of vanillyl fatty acid esters from salmon oil in a solvent-free medium

This study hypothesizes that the solvent-free alcoholysis of oil recovered from salmon heads using vanillyl alcohol (VA) and immobilized lipase B can efficiently produce esters with enhanced stability and antioxidant properties. The objective was to investigate the selectivity and resulting ester profile, which may provide nutritional and functional advantages compared to supplementing oil with vanillyl alcohol. After 24 h, nearly complete conversion of vanillyl alcohol was achieved, leading to the production of various esters reflective of the oil's original fatty acid composition. The synthesis of esters like oleoyl and linolenoyl was favored over docosahexaenoyl and linoleoyl esters, influenced by fatty acid distribution and enzyme specificity, along with potential intra-molecular acyl transfer isomerization. The reaction medium demonstrated significant stability and antioxidant activity, highlighting the potential benefits of vanillyl esters over traditional supplementation methods. These findings suggest that the phenolic alcohol-based alcoholysis of fish oil offers a promising approach to generating stable, nutritionally valuable extracts with potent antioxidant capabilities.

Advanced design of target-driven self-powered sensor assisted by cascade catalytic strategy

In this work, a self-powered microsensor platform based on enzyme biofuel cells (EBFCs) was developed for intelligent monitoring of disease markers miRNA-451. The cascade catalysis system constructed by using the strategy of enzyme-like ZIF-8 nanocapsule incorporation with biological enzymes, which could simultaneously take into account the specificity of biological enzymes and the high activity of nano-enzymes, significantly promoted the electron transfer between glucose and the bio-anode surface, and improved the sensitivity and stability of the sensing system. Meanwhile, the target-triggered hybridisation chain reaction (HCR) amplification strategy to achieve exponential signal amplification based on accurate recognition, and jointly improve the detection sensitivity. As expected, the micro-sensor platform has a wide linear range of 0.5-1.0 fmol/L with a low limit of detection (LOD) of 0.13 fmol/L (S/N=3) and exhibits excellent selectivity, reproducibility and stability in interference assays under optimal detection conditions. The designed self-powered system is simple to construct, easy to transport and the data transmission mode is intelligent and controllable, which is expected to be used in basic biochemical research, clinical diagnosis and environmental monitoring.

Feature sequence-based genome mining uncovers the hidden diversity of bacterial siderophore pathways.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

Surface-engineered bacteria in drug development.

Bacterial surface display in combination with fluorescence-activated cell sorting is a versatile and robust system and an interesting alternative approach to phage display for the generation of therapeutic affinity proteins. The system enables real-time monitoring and sorting of cell populations, which presents unique possibilities for drug development. It has been used to develop several affibody molecules currently being evaluated preclinically for the treatment and diagnosis of, for example, cancer and neurodegenerative diseases. Additionally, it can be implemented in other areas of drug design, such as for mapping epitopes and evolving enzyme specificities.

Elucidation of a specific alginate lyase Aly7Rm: The products demonstrated the strict recognition of G residue at subsites ±2

An alginate lyase Aly7Rm with novel subsite specificity was cloned, expressed and characterized in this article. Aly7Rm was a G-specific alginate lyase, exhibiting optimum reaction conditions at 30 °C and pH 5.0. UPSEC-VWD-MS analysis revealed that Aly7Rm degraded polysaccharides in a random endo-acting manner. The main degradation products of alginate were trisaccharide to octasaccharide and those of PG were disaccharide to hexasaccharide. The product distribution was related to the specificity of enzyme and the structure of substrates. By utilizing the HPAEC-PAD/MS method, the structure of products was identified and the specificities of subsite were defined. Aly7Rm accommodated both G and M at subsites −1 and strictly recognized G at subsite −2 and +2. This is the first report on the alginate lyase exhibiting G specificity at subsites ±2, which shed light on the diversity in subsite specificity of alginate lyases. The Aly7Rm with clear action pattern could serve as an efficient tool in the specific degradation of alginate and targeted preparation of oligosaccharides.

Substrate-Multiplexed Assessment of Aromatic Prenyltransferase Activity.

An increasingly effective strategy to identify synthetically useful enzymes is to sample the diversity already present in Nature. Here, we construct and assay a panel of phylogenetically diverse aromatic prenyltransferases (PTs). These enzymes catalyze a variety of C-C bond forming reactions in natural product biosynthesis and are emerging as tools for synthetic chemistry and biology. Homolog screening was further empowered through substrate-multiplexed screening, which provides direct information on enzyme specificity. We perform a head-to-head assessment of the model members of the PT family and further identify homologs with divergent sequences that rival these superb enzymes. This effort revealed the first bacterial O-Tyr PT and, together, provide valuable benchmarking for future synthetic applications of PTs.

Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from Geobacillus kaustophilus.

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The GkHAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that GkHAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global GkHAL folding. These findings suggest that GkHAL could be a promising candidate for potential biotechnological applications.

Substrate specificity of a branch of aromatic dioxygenases determined by three distinct motifs

The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.

The INSIGHT platform: Enhancing NAD(P)-dependent specificity prediction for co-factor specificity engineering

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR.

Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols. An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time. This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.

Bacterial Purine Nucleoside Phosphorylases from Mesophilic and Thermophilic Sources: Characterization of Their Interaction with Natural Nucleosides and Modified Arabinofuranoside Analogues.

The enzymatic synthesis of nucleoside derivatives is an important alternative to multi-step chemical methods traditionally used for this purpose. Despite several undeniable advantages of the enzymatic approach, there are a number of factors limiting its application, such as the limited substrate specificity of enzymes, the need to work at fairly low concentrations, and the physicochemical properties of substrates-for example, low solubility. This research conducted by our group is dedicated to the advantages and limitations of using purine nucleoside phosphorylases (PNPs), the main enzymes for the metabolic reutilization of purines, in the synthesis of modified nucleoside analogues. In our work, the substrate specificity of PNP from various bacterial sources (mesophilic and thermophilic) was studied, and the effect of substrate, increased temperature, and the presence of organic solvents on the conversion rate was investigated.

Chemoenzymatic Synthesis of Plant‐Derived Kavalactone Natural Products by Dynamic Resolution Using a Biosynthetic O‐Methyltransferase Tailoring Enzyme

Biosynthetic enzymes have enormous potential for the chemoenzymatic synthesis of natural products and other bioactive compounds. Methyltransferases are promising tools for the selective enzymatic modification of complex structures. This paper describes the production, purification and biochemical characterization of the O‐methyltransferase JerF, which catalyzes unique 4‐methoxy‐5,6‐dihydropyranone formation in jerangolid A biosynthesis. Isolation problems had hitherto prevented detailed studies on JerF and were solved by the production of the maltose‐binding protein fusion protein. The differentiation of JerF between styryl‐substituted dihydropyrandion enantiomers was investigated. In combination with a spontaneous racemization occurring with this type of substrates, a new enzymatic dynamic kinetic resolution was observed, which was used for the enantioselective chemoenzymatic synthesis of kavalactone natural products and new derivatives. In combination with an HMT‐based SAM regeneration system, (+)‐kavain, (+)‐11,12‐dimethoxykavain and (+)‐12‐fluorokavain were prepared in 3‐4 steps on the 100 µmol scale with overall yields of 37‐57% and ees of 70‐86%. A mutational study based on an AlphaFold 2 model provided indications for active site residues with an influence on the performance of the enzyme that could be targets for engineering. This example illustrates how the exceptional enzymatic activities and specificities of biosynthetic enzymes can be exploited for the development of new synthesis approaches.

Enzyme engineering in microbial biosynthesis of terpenoids: progress and perspectives

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-seq.

End-to-end RNA-sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RTs used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those derived from group II introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multistep template-switching reaction carried out by RTs, in this case, by MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized the nucleotide specificity of nontemplated addition (NTA) reaction that occurs when the RT extends past the RNA 5'-terminus. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template-switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq from total human RNA and poly(A)-enriched RNA, with short- and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.

A novel cisAB allele with a missense variant (c.971T>C) in the ABO gene of a Brazilian family.

Missense variants in exon 7 of the ABO gene can lead to the formation of cisAB alleles. These alleles encode glycosyltransferases (GTs) capable of synthesizing both A and B antigens. In this study, we report the discovery of a novel cisAB allele and characterize it at molecular, protein and serological levels. Blood and DNA samples from the proband and seven relatives were examined using standard and modified ABO phenotyping, polymerase chain reaction-restriction fragment length polymorphism and ABO gene sequencing. We assessed the impact of the p.Leu324Ser variant on the protein structure of the mutant GT using bioinformatics tools. Molecular tests revealed a c.971T>C (p.Leu324Ser) variant in the ABO gene in five of the eight individuals. This variant results in a GT that produces more A antigens and fewer B antigens. Bioinformatics analysis suggests that the amino acid substitution (p.Leu324Ser) could potentially affect enzymatic activity and specificity of the GT. We identified a novel cisAB allele resulting from a c.971T>C variant in the ABO gene. This variant led to the expression of an ABweak phenotype.