Probing the enzymatic activity and maturation process of the EcAIII Ntn-amidohydrolase using local random mutagenesis.

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.

Hydrophobic substrate binding pocket remodeling of echinocandin B deacylase based on multi-dimensional rational design.

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates.

Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

Toward More Selective Antibiotic Inhibitors: A Structural View of the Complexed Binding Pocket of E. coli Peptide Deformylase.

Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

LpxC inhibition: Potential and opportunities with carbohydrate scaffolds.

Uridine diphosphate-3-O-(hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a key enzyme involved in the biosynthesis of lipid A, an essential building block, for the construction and assembly of the outer membrane (OM) of Gram-negative bacteria. The enzyme is highly conserved in almost all Gram-negative bacteria and hence has emerged as a promising target for drug discovery in the fight against multi-drug resistant Gram-negative infections. Since the first nanomolar LpxC inhibitor, L-161,240, an oxazoline-based hydroxamate, the two-decade-long ongoing search has provided valuable information regarding essential features necessary for inhibition. Although the design and structure optimization for arriving at the most efficacious inhibitor of this enzyme has made good use of different heterocyclic moieties, the use of carbohydrate scaffold is scant. This review briefly covers the advancement and progress made in LpxC inhibition. The field awaits the use of potential associated with carbohydrate-based scaffolds for LpxC inhibition and the discovery of anti-bacterial agents against Gram-negative infections.

Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.

The complexed crystal structure of dihydropyrimidinase reveals a potential interactive link with the neurotransmitter γ-aminobutyric acid (GABA).

Dihydropyrimidinase (DHPase) plays a crucial role in pyrimidine degradation, showcasing a broad substrate specificity that extends beyond pyrimidine catabolism, hinting at additional roles for this ancient enzyme. In this study, we solved the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with the neurotransmitter γ-aminobutyric acid (GABA) at a resolution of 1.97 Å (PDB ID 8WQ9). Our structural analysis revealed two GABA binding sites in each monomer of PaDHPase. Interactions between PaDHPase and GABA molecules, involving residues within a contact distance of <4 Å, were examined. In silico analyses via PISA and PLIP software revealed hydrogen bonds formed between the side chain of Cys318 and GABA 1, as well as the main chains of Ser333, Ile335, and Asn337 with GABA 2. Comparative structural analysis between GABA-bound and unbound states unveiled significant conformational changes at the active site, particularly within dynamic loop I, supporting the conclusion that PaDHPase binds GABA through the loop-out mechanism. Building upon this molecular evidence, we discuss and propose a working model. The study expands the GABA interactome by identifying DHPase as a novel GABA-interacting protein and provides structural insight into the interaction between a dimetal center in the protein's active site and GABA. Further investigations are warranted to explore potential interactions of GABA with other DHPase-like proteins and to understand whether DHPase may have additional regulatory and physiological roles in the cell, extending beyond pyrimidine catabolism.

Structural Insights into the Catalytic Activity of Cyclobacterium marinum N-Acetylglucosamine Deacetylase.

N-Acetylglucosamine deacetylase from Cyclobacterium marinum (CmCBDA) is a highly effective and selective biocatalyst for the production of d-glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). However, the underlying catalytic mechanism remains elusive. Here, we show that CmCBDA is a metalloenzyme with a preference for Ni2+ over Mn2+. Crystal structures of CmCBDA in complex with Ni2+ and Mn2+ revealed slight remodeling of the CmCBDA active site by the metal ions. We also demonstrate that CmCBDA exists as a mixture of homodimers and monomers in solution, and dimerization is indispensable for catalytic activity. A mutagenesis analysis also indicated that the active site residues Asp22, His72, and His143 as well as the residues involved in dimerization, Pro52, Trp53, and Tyr55, are essential for catalytic activity. Furthermore, a mutation on the protein surface, Lys219Glu, resulted in a 2.3-fold improvement in the deacetylation activity toward GlcNAc. Mechanistic insights obtained here may facilitate the development of CmCBDA variants with higher activities.

Biochemical Characterization of Thermostable Acrylamide Amidohydrolase from Aspergillus fumigatus with Potential Activity for Acrylamide Degradation in Various Food Products.

Acrylamide is the major by-product of the Maillard reactions in foods with the overheating processes of L-asparagine-rich foods with reducing sugars that usually allied with neurotoxicity and carcinogenicity. Several approaches have been used to prevent the formation of acrylamide, however, degrading the already formed acrylamide in foods remains unequivocal. Acrylamide hydrolyzing enzyme "amidohydrolase" is one of the most promising enzymes for acrylamide degradation in foods. So, amidohydrolase "amidase" from thermotolerant Aspergillus fumigatus EFBL was purified to their electrophoretic homogeneity by gel-filtration and ion-exchange chromatography, with overall purification folds 2.8 and yield 9.43%. The apparent molecular subunit structure of the purified A. fumigatus amidase was 50 kDa, with highest activity at reaction temperature of 40 °C and pH of 7.5 The enzyme displayed a significant thermal stability as revealed from the value of T1/2 (13.37 h), and thermal denaturation rate (Kr 0.832 × 10-3 min) at 50 °C, with metalloproteinic identity. The purified enzyme had a significant activity for acrylamide degradation in various food products such as meat, cookies, potato chips, and bread as revealed from the HPLC analysis and LC-MS analysis. So, with the purified amidase, the acrylamide in the food products was degraded by about 95% to acrylic acid, ensuring the possibility of using this enzyme in abolishing the toxic acrylamide in the foods products. This is the first report exploring the potency of A. fumigatus amidase for an actual degradation of acrylamide in foods efficiently. Further biochemical analyses are ongoing to assess the affinity of this enzyme for selective hydrolyses of acrylamide in foods, without affecting the beneficial stereochemical related compounds.

Bacterial Zinc Metalloenzyme Inhibitors: Recent Advances and Future Perspectives.

Human deaths caused by Gram-negative bacteria keep rising due to the multidrug resistance (MDR) phenomenon. Therefore, it is a priority to develop novel antibiotics with different mechanisms of action. Several bacterial zinc metalloenzymes are becoming attractive targets since they do not show any similarities with the human endogenous zinc-metalloproteinases. In the last decades, there has been an increasing interest from both industry and academia in developing new inhibitors against those enzymes involved in lipid A biosynthesis, and bacteria nutrition and sporulation, e.g., UDP-[3-O-(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), thermolysin (TLN), and pseudolysin (PLN). Nevertheless, targeting these bacterial enzymes is harder than expected and the lack of good clinical candidates suggests that more effort is needed. This review gives an overview of bacterial zinc metalloenzyme inhibitors that have been synthesized so far, highlighting the structural features essential for inhibitory activity and the structure-activity relationships. Our discussion may stimulate and help further studies on bacterial zinc metalloenzyme inhibitors as possible novel antibacterial drugs.

Deformylation of nascent peptide chains on the ribosome.

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.

Ring-Closure on the Rocks in a Prebiotic Environment.

Ring-closure is a key step in current pyrimidine anabolism and one may wonder whether cyclisation reactions could be promoted in the geochemical context at the origins of life, i. e. with the help of minerals. Various prebiotic minerals were tested in this work, including silica, carbonates, microporous minerals. In particular, the role of zinc ions supported on minerals was investigated in view of its presence in the catalytic site of cyclic amidohydrolase enzymes. Based on in situ (TGA: ThermoGravimetric Analysis, ATR-IR: Attenuated Total Reflectance-InfraRed) and ex situ (1 H NMR- Nuclear Magnetic Resonance) characterisations, we identified the products of thermal activation of NCA (N-carbamoyl-aspartic acid) in wetting-and-drying scenarios on the surface of minerals. NCA can cyclize extensively only on some surfaces, with the predominant product being 5-carboxymethylhydantoin (Hy) rather than dihydroorotate (DHO), while there is a competition with hydrolysis on others. Replacing the enzymes with heterogeneous catalysts also works with other reactions catalysed by enzymes of the cyclic amidohydrolases family. The role of the hydrophilicity/hydrophobicity of minerals as well as the regioselectivity of the cyclisation (5-carboxymethylhydantoin versus dihydroorotate) are examined.

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses.

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase.

Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of ß-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding N-carbamoyl-ß-amino acids. This enzyme has the potential to be used as a tool in the production of ß-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from Saccharomyces kluyveri and Sinorhizobium meliloti CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol-1. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.

Experimental and computational approaches to characterize a novel amidase that initiates the biodegradation of the herbicide propanil in Bosea sp. P5.

The herbicide propanil and its major metabolite 3,4-dichloroaniline (3,4-DCA) are difficult to biodegrade and pose great health and environmental risks. However, studies on the sole or synergistic mineralization of propanil by pure cultured strains are limited. A two-strain consortium (Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34), obtained from a swep-mineralizing enrichment culture that can synergistically mineralize propanil, has been previously reported. Here, another propanil degradation strain, Bosea sp. P5, was successfully isolated from the same enrichment culture. A novel amidase, PsaA, responsible for initial propanil degradation, was identified from strain P5. PsaA shared low sequence identity (24.0-39.7 %) with other biochemically characterized amidases. PsaA exhibited optimal activity at 30 °C and pH 7.5 and had kcat and Km values of 5.7 s-1 and 125 µM, respectively. PsaA could convert the herbicide propanil to 3,4-DCA but exhibited no activity toward other herbicide structural analogs. This catalytic specificity was explained by using propanil and swep as substrates and then analyzed by molecular docking, molecular dynamics simulation and thermodynamic calculations, which revealed that Tyr138 is the key residue that affects the substrate spectrum of PsaA. This is the first propanil amidase with a narrow substrate spectrum identified, providing new insights into the catalytic mechanism of amidase in propanil hydrolysis.

Structural and Functional Characterization of the Ureidoacrylate Amidohydrolase RutB from Escherichia coli.

We present a detailed structure-function analysis of the ureidoacrylate amidohydrolase RutB from Eschericha coli, which is an essential enzyme of the Rut pathway for pyrimidine utilization. Crystals of selenomethionine-labeled RutB were produced, which allowed us to determine the first structure of the enzyme at a resolution of 1.9 Å and to identify it as a new member of the isochorismatase-like hydrolase family. RutB was co-crystallized with the substrate analogue ureidopropionate, revealing the mode of substrate binding. Mutation of residues constituting the catalytic triad (D24A, D24N, K133A, C166A, C166S, C166T, C166Y) resulted in complete inactivation of RutB, whereas mutation of other residues close to the active site (Y29F, Y35F, N72A, W74A, W74F, E80A, E80D, S92A, S92T, S92Y, Q105A, Y136A, Y136F) leads to distinct changes of the turnover number (kcat) and/or the Michaelis constant (KM). The results of our structural and mutational studies allowed us to assign specific functions to individual residues and to formulate a plausible reaction mechanism for RutB.

Crystal Structure of Allantoinase from Escherichia coli BL21: A Molecular Insight into a Role of the Active Site Loops in Catalysis

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.

Engineering the Thermostability of a d-Carbamoylase Based on Ancestral Sequence Reconstruction for the Efficient Synthesis of d-Tryptophan.

Employing ancestral sequence reconstruction and consensus sequence analysis, the thermostability of a novel d-carbamoylase derived from Nitratireductor indicus (NiHyuC) was engineered through greedy-oriented iterative combinatorial mutagenesis. A mutant S202P/E208D/R277L (M4Th3) was obtained with significantly elevated thermostability. M4Th3 has a half-life of 36.5 h at 40 °C, about 28.5 times of 1.3 h of its parent M4. For the reaction at 40 °C, M4Th3 can catalyze 10 mM N-carbamoyl-d-tryptophan to produce d-tryptophan with a conversion ratio of 96.4% after 12 h, which is significantly higher than 64.1% of M4. MD simulation reveals that new hydrogen bonds emerging from E208D on the surface can increase the hydrophobicity of the protein, leading to improved stability. More importantly, R277L could contribute to enhanced interface stability of homodimeric M4. This study provides a thermostable d-carbamoylase for the "hydantoinase process", which has potential in the industrial synthesis of optically pure natural and non-natural amino acids.