Enhancing uric acid electrochemical detection with copper ion-activated mini protein mimicking uricase within ZIF-8: response surface methodology (RSM) optimization.

This study aims to investigate the influence of copper(II) ions as a cofactor on the electrochemical performance of a biocomposite consisting of a mini protein mimicking uricase (mp20) and zeolitic immidazolate framework-8 (ZIF-8) for the detection of uric acid. A central composite design (CCD) was utilized to optimize the independent investigation, including pH, deposition potential, and deposition time, while the current response resulting from the electrocatalytic oxidation of uric acid was used as the response. The statistical analysis of variance (ANOVA) showed a good correlation between the experimental and predicted data, with a residual standard error percentage (RSE%) of less than 2% for predicting optimal conditions. The synergistic effect of the nanoporous ZIF-8 host, Cu(II)-activated mp20, and reduced graphene oxide (rGO) layer resulted in a highly sensitive biosensor with a limit of detection (LOD) of 0.21 µM and a reproducibility of the response (RSD = 0.63%). The Cu(II)-activated mp20@ZIF-8/rGO/SPCE was highly selective in the presence of common interferents, and the fabricated layer exhibited remarkable stability with signal changes below 4.15% after 60 days. The biosensor's reliable performance was confirmed through real sample analyses of human serum and urine, with comparable recovery values to conventional HPLC.

Rational design of mini protein mimicking uricase: Encapsulation in ZIF-8 for uric acid detection.

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction.

Knotting terminal ends of mutant T1 lipase with disulfide bond improved structure rigidity and stability.

Lipase biocatalysts offer unique properties which are often impaired by low thermal and methanol stability. In this study, the rational design was employed to engineer a disulfide bond in the protein structure of Geobacillus zalihae T1 lipase in order to improve its stability. The selection of targeted disulfide bond sites was based on analysis of protein spatial configuration and change of Gibbs free energy. Two mutation points (S2C and A384C) were generated to rigidify the N-terminal and C-terminal regions of T1 lipase. The results showed the mutant 2DC lipase improved methanol stability from 35 to 40% (v/v) after 30 min of pre-incubation. Enhancement in thermostability for the mutant 2DC lipase at 70 °C and 75 °C showed higher half-life at 70 °C and 75 °C for 30 min and 52 min, respectively. The mutant 2DC lipase maintained the same optimum temperature (70 °C) as T1 lipase, while thermally induced unfolding showed the mutant maintained higher rigidity. The kcat/Km values demonstrated a relatively small difference between the T1 lipase (WT) and 2DC lipase (mutant). The kcat/Km (s-1 mM-1) of the T1 and 2DC showed values of 13,043 ± 224 and 13,047 ± 312, respectively. X-ray diffraction of 2DC lipase crystal structure with a resolution of 2.04 Å revealed that the introduced single disulfide bond did not lower initial structural interactions within the residues. Enhanced methanol and thermal stability are suggested to be strongly related to the newly disulfide bridge formation and the enhanced compactness and rigidity of the mutant structure. KEY POINTS: • Protein engineering via rational design revealed relative improved enzymatic performance. • The presence of disulfide bond impacts on the rigidity and structural function of proteins. • X-ray crystallography reveals structural changes accompanying protein modification.

Protective effect of Clinacanthus nutans in cisplatin-induced nephrotoxicity on human kidney cell (PCS-400-010) elucidated by an LCMS-based metabolomics approach.

Cisplatin-induced nephrotoxicity has been widely reported in numerous studies. The objective of this study is to assess the potential nephroprotective effects of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae) leaf extracts on human kidney cells (PCS-400-010) in vitro using an LCMS-based metabolomics approach. Orthogonal partial least square-discriminant analysis identified 16 significantly altered metabolites when comparing the control and pre-treated C. nutans cisplatin-induced groups. These metabolites were found to be associated with glycerophospholipid, purine, and amino acid metabolism, as well as the glycolysis pathway. Pre-treatment with C. nutans aqueous extract (125 µg/mL) for 24 h, followed by 48 h of cisplatin induction in PCS-400-010 cells, demonstrated a nephroprotective effect, particularly involving the regulation of amino acid metabolism.

Thermostability engineering of industrial enzymes through structure modification.

Thermostability is an essential requirement of enzymes in the industrial processes to catalyze the reactions at high temperatures; thus, enzyme engineering through directed evolution, semi-rational design and rational design are commonly employed to construct desired thermostable mutants. Several strategies are implemented to fulfill enzymes' thermostability demand including decreasing the entropy of the unfolded state through substitutions Gly → Xxx or Xxx → Pro, hydrogen bond, salt bridge, introducing two different simultaneous interactions through single mutant, hydrophobic interaction, filling the hydrophobic cavity core, decreasing surface hydrophobicity, truncating loop, aromatic-aromatic interaction and introducing positively charged residues to enzyme surface. In the current review, horizons about compatibility between secondary structures and substitutions at preferable structural positions to generate the most desirable thermostability in industrial enzymes are broadened. KEY POINTS: • Protein engineering is a powerful tool for generating thermostable industrial enzymes. • Directed evolution and rational design are practical approaches in enzyme engineering. • Substitutions in preferable structural positions can increase thermostability.

Feather-Degrading Bacillus cereus HD1: Genomic Analysis and Its Optimization for Keratinase Production and Feather Degradation.

Keratinase is an important enzyme that is used to degrade feather wastes produced by poultry industries and slaughterhouses that accumulate rapidly over time. The search for keratinase-producing microorganisms is important to potentially substitute physicochemical treatments of feather waste. In this study, the genome of Bacillus cereus HD1 and its keratinolytic prowess was investigated. The whole-genome shotgun size is 5,668,864 bp consisting of 6083 genes, 69 tRNAs, and 10 rRNAs. The genomic analyses revealed 15 potential keratinase genes and other enzymes that might assist keratin degradation, such as disulfide reductase and cysteine dioxygenase. The optimal conditions for feather degradation and keratinase production by B. cereus HD1 such as incubation time, pH, temperature, yeast extract, and glycerol concentrations were determined to be 5 days, pH 8, 37 °C, 0.05% (w/v), and 0.1% (v/v), respectively. Under optimized conditions, B. cereus HD1 exhibited feather degradation of 65%, with bacterial growth and maximum keratinase activity of 1.3 × 1011 CFU/mL and 41 U/mL, respectively, after 5 days of incubation in a feather basal medium. The findings obtained from this study may facilitate further research into utilizing B. cereus HD1 as a prominent keratinolytic enzymes production host and warrant potential biotechnological applications.

An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation.

Plastic or microplastic pollution is a global threat affecting ecosystems, with the current generation reaching as much as 400 metric tons per/year. Soil ecosystems comprising agricultural lands act as microplastics sinks, though the impact could be unexpectedly more far-reaching. This is troubling as most plastic forms, such as polyethylene terephthalate (PET), formed from polymerized terephthalic acid (TPA) and ethylene glycol (EG) monomers, are non-biodegradable environmental pollutants. The current approach to use mechanical, thermal, and chemical-based treatments to reduce PET waste remains cost-prohibitive and could potentially produce toxic secondary pollutants. Thus, better remediation methods must be developed to deal with plastic pollutants in marine and terrestrial environments. Enzymatic treatments could be a plausible avenue to overcome plastic pollutants, given the near-ambient conditions under which enzymes function without the need for chemicals. The discovery of several PET hydrolases, along with further modification of the enzymes, has considerably aided efforts to improve their ability to degrade the ester bond of PET. Hence, this review emphasizes PET-degrading microbial hydrolases and their contribution to alleviating environmental microplastics. Information on the molecular and degradation mechanisms of PET is also highlighted in this review, which might be useful in the future rational engineering of PET-hydrolyzing enzymes.

Thermostable lipases and their dynamics of improved enzymatic properties.

Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.

Molecular strategies to increase keratinase production in heterologous expression systems for industrial applications.

Keratinase is an important enzyme that can degrade recalcitrant keratinous wastes to form beneficial recyclable keratin hydrolysates. Keratinase is not only important as an alternative to reduce environmental pollution caused by chemical treatments of keratinous wastes, but it also has industrial significance. Currently, the bioproduction of keratinase from native keratinolytic host is considered low, and this hampers large-scale usage of the enzyme. Straightforward approaches of cloning and expression of recombinant keratinases from native keratinolytic host are employed to elevate the amount of keratinase produced. However, this is still insufficient to compensate for the lack of its large-scale production to meet the industrial demands. Hence, this review aimed to highlight the various sources of keratinase and the strategies to increase its production in native keratinolytic hosts. Molecular strategies to increase the production of recombinant keratinase such as plasmid selection, promoter engineering, chromosomal integration, signal peptide and propeptide engineering, codon optimization, and glycoengineering were also described. These mentioned strategies have been utilized in heterologous expression hosts, namely, Escherichia coli, Bacillus sp., and Pichia pastoris, as they are most widely used for the heterologous propagations of keratinases to further intensify the production of recombinant keratinases adapted to better suit the large-scale demand for them. KEY POINTS: • Molecular strategies to enhance keratinase production in heterologous hosts. • Construction of a prominent keratinolytic host from a native strain. • Patent analysis of keratinase production shows rapid high interest in molecular field.

Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-ß-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-ß-lactamase.

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-ß-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications.

The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases­key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 µM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources.

A Sco protein among the hypothetical proteins of Bacillus lehensis G1: Its 3D macromolecular structure and association with Cytochrome C Oxidase.

BACKGROUND: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail. RESULTS: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the ßαßαßß core structure of Trx-like proteins as well as three flanking ß-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes. CONCLUSIONS: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.

Structure to function prediction of hypothetical protein KPN_00953 (Ycbk) from Klebsiella pneumoniae MGH 78578 highlights possible role in cell wall metabolism.

BACKGROUND: Klebsiella pneumoniae plays a major role in causing nosocomial infection in immunocompromised patients. Medical inflictions by the pathogen can range from respiratory and urinary tract infections, septicemia and primarily, pneumonia. As more K. pneumoniae strains are becoming highly resistant to various antibiotics, treatment of this bacterium has been rendered more difficult. This situation, as a consequence, poses a threat to public health. Hence, identification of possible novel drug targets against this opportunistic pathogen need to be undertaken. In the complete genome sequence of K. pneumoniae MGH 78578, approximately one-fourth of the genome encodes for hypothetical proteins (HPs). Due to their low homology and relatedness to other known proteins, HPs may serve as potential, new drug targets. RESULTS: Sequence analysis on the HPs of K. pneumoniae MGH 78578 revealed that a particular HP termed KPN_00953 (YcbK) contains a M15_3 peptidases superfamily conserved domain. Some members of this superfamily are metalloproteases which are involved in cell wall metabolism. BLASTP similarity search on KPN_00953 (YcbK) revealed that majority of the hits were hypothetical proteins although two of the hits suggested that it may be a lipoprotein or related to twin-arginine translocation (Tat) pathway important for transport of proteins to the cell membrane and periplasmic space. As lipoproteins and other components of the cell wall are important pathogenic factors, homology modeling of KPN_00953 was attempted to predict the structure and function of this protein. Three-dimensional model of the protein showed that its secondary structure topology and active site are similar with those found among metalloproteases where two His residues, namely His169 and His209 and an Asp residue, Asp176 in KPN_00953 were found to be Zn-chelating residues. Interestingly, induced expression of the cloned KPN_00953 gene in lipoprotein-deficient E. coli JE5505 resulted in smoother cells with flattened edges. Some cells showed deposits of film-like material under scanning electron microscope. CONCLUSIONS: We postulate that KPN_00953 is a Zn metalloprotease and may play a role in bacterial cell wall metabolism. Structural biology studies to understand its structure, function and mechanism of action pose the possibility of utilizing this protein as a new drug target against K. pneumoniae in the future.

Insight on recently discovered PET polyester-degrading enzymes, thermostability and activity analyses.

The flexibility and the low production costs offered by plastics have made them crucial to society. Unfortunately, due to their resistance to biological degradation, plastics remain in the environment for an extended period of time, posing a growing risk to life on earth. Synthetic treatments of plastic waste damage the environment and may cause damage to human health. Bacterial and fungal isolates have been reported to degrade plastic polymers in a logistic safe approach with the help of their microbial cell enzymes. Recently, the bacterial strain Ideonella sakaiensis (201-F6) was discovered to break down and assimilate polyethylene terephthalate (PET) plastic via metabolic processes at 30 °C to 37 °C. PETase and MHETase enzymes help the bacterium to accomplish such tremendous action at lower temperatures than previously discovered enzymes. In addition to functioning at low temperatures, the noble bacterium's enzymes have amazing qualities over pH and PET plastic degradation, including a shorter period of degradation. It has been proven that using the enzyme PETase, this bacterium hydrolyzes the ester linkages of PET plastic, resulting in production of terephthalic acid (TPA), nontoxic compound and mono-2-hydroxyethyl (MHET), along with further depolymerization of MHET to release ethylene glycogen (EG) and terephthalic acid (TPA) by the second enzyme MHETase. Enzymatic plastic degradation has been proposed as an environmentally friendly and long-term solution to plastic waste in the environment. As a result, this review focuses on the enzymes involved in hydrolyzing PET plastic polymers, as well as some of the other microorganisms involved in plastic degradation.

Bioinspired mp20 mimicking uricase in ZIF-8: Metal ion dependent for controllable activity.

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 µM) with a low limit (4.4 µM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications.

Various polar tripeptides as asymmetric organocatalyst in direct aldol reactions in aqueous media.

A series of tripeptide organocatalysts containing a secondary amine group and two amino acids with polar side chain units were developed and evaluated in the direct asymmetric intermolecular aldol reaction of 4-nitrobenzaldehyde and cyclohexanone. The effectiveness of short polar peptides as asymmetric catalysts in aldol reactions to attain high yields of enantio- and diastereoselective isomers were investigated. In a comparison, glutamic acid and histidine produced higher % ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric aldol addition reaction in aqueous media.

Probing the substrate binding modes and catalytic mechanisms of BLEG-1, a promiscuous B3 metallo-ß-lactamase with glyoxalase II properties.

BLEG-1 from Bacillus lehensis G1 is an evolutionary divergent B3 metallo-ß-lactamase (MBL) that exhibited both ß-lactamase and glyoxalase II (GLXII) activities. Sequence, phylogeny, biochemical and structural relatedness of BLEG-1 to B3 MBL and GLXII suggested BLEG-1 might be an intermediate in the evolutionary path of B3 MBL from GLXII. The unique active site cavity of BLEG-1 that recognizes both ß-lactam antibiotics and S-D-lactoylglutathione (SLG) had been postulated as the key factor for its dual activity. In this study, dynamic ensembles of BLEG-1 and its substrate complexes divulged conformational plasticity and binding modes of structurally distinct substrates to the enzyme, providing better insights into its structure-to-function relationship and enzymatic promiscuity. Our results highlight the flexible nature of the active site pocket of BLEG-1, which is governed by concerted loop motions involving loop7+α3+loop8 and loop12 around the catalytic core, thereby moulding the binding pocket and facilitate interactions of BLEG-1 with both ampicillin and SLG. The distribution of (i) predominantly hydrophobic amino acids in the N-terminal domain, and (ii) flexible amino acids with polar and/or charged side chains in both N- and C-termini provide additional advantages to BLEG-1 in confining the aromatic group of ampicillin, and polar groups of SLG, respectively. The importance of these residues for substrates binding was further confirmed by the reduction in MBL and GLXII activities upon alanine substitutions of Ile-10, Phe-57, Arg-94, Leu-95, and Arg-159. Based on molecular dynamics simulation, mutational, and biochemical data presented herein, the catalytic mechanisms of BLEG-1 toward the hydrolysis of ß-lactams and SLG were proposed.