Biodegradation of ochratoxin A by Brevundimonas diminuta HAU429: Characterized performance, toxicity evaluation and functional enzymes.

Ochratoxin A (OTA) is a notorious mycotoxin commonly contaminating food products worldwide. In this study, an OTA-degrading strain Brevundimonas diminuta HAU429 was isolated by using hippuryl-L-phenylalanine as the sole carbon source. The biodegradation of OTA by strain HAU429 was a synergistic effect of intracellular and extracellular enzymes, which transformed OTA into ochratoxin α (OTα) through peptide bond cleavage. Cytotoxicity tests and cell metabolomics confirmed that the transformation of OTA into OTα resulted in the detoxification of its hepatotoxicity since OTA but not OTα disturbed redox homeostasis and induced oxidative damage to hepatocytes. Genome mining identified nine OTA hydrolase candidates in strain HAU429. They were heterologously expressed in Escherichia coli, and three novel amidohydrolase BT6, BT7 and BT9 were found to display OTA-hydrolyzing activity. BT6, BT7 and BT9 showed less than 45 % sequence identity with previously identified OTA-degrading amidohydrolases. BT6 and BT7 shared 60.9 % amino acid sequence identity, and exhibited much higher activity towards OTA than BT9. BT6 and BT7 could completely degrade 1 µg mL-1 of OTA within 1 h and 50 min, while BT9 hydrolyzed 100 % of OTA in the reaction mixture by 12 h. BT6 was the most thermostable retaining 38 % of activity after incubation at 70 °C for 10 min, while BT7 displayed the highest tolerance to ethanal remaining 76 % of activity in the presence of 6 % ethanol. This study could provide new insights towards microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification during food processing.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes.

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

A novel N-acetylglucosamine-6-phosphate deacetylase that is essential for chitin utilization in the chitinolytic bacterium, Chitiniphilus shinanonensis.

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.

Transcriptomic signature, bioactivity and safety of a non-hepatotoxic analgesic generating AM404 in the midbrain PAG region.

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids' addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund's adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001's safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

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.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase.

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (- 80, - 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including - 80, - 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

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.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

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.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Asp-tRNAAsn/Glu-tRNAGln amidotransferase A subunit-like amidase mediates the degradation of insecticide flonicamid by Variovorax boronicumulans CGMCC 4969.

The main metabolic product of the pyridinecarboxamide insecticide flonicamid, N-(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product N-(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium Variovorax boronicumulans CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by V. boronicumulans CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding amiD was cloned and expressed recombinantly in Escherichia coli, and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic L-phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in V. boronicumulans CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the amiD gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation.

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.

Depolymerization of the polyester-polyurethane by amidase GatA250 and enhancing the production of 4,4'-methylenedianiline with cutinase LCC.

Polyurethane (PU) is a complex polymer synthesized from polyols and isocyanates. It contains urethane bonds that resist hydrolysis, which decreases the efficiency of biodegradation. In this study, we first expressed the amidase GatA250, and then, assessed the enzymatic characterization of GatA250 and its efficiency in degrading the polyester-PU. GatA250 degraded self-synthesized thermoplastic PU film and postconsumption foam with degradation efficiency of 8.17% and 4.29%, respectively. During the degradation, the film released 14.8 µm 4,4'-methylenedianiline (MDA), but 1,4-butanediol (BDO) and adipic acid (AA) were not released. Our findings indicated that GatA250 only cleaved urethane bonds in PU, and the degradation efficiency was extremely low. Hence, we introduced the cutinase LCC, which possesses hydrolytic activity on the ester bonds in PU, and then used both enzymes simultaneously to degrade the polyester-PU. The combined system (LCC-GatA250) had higher degradation efficiency for the degradation of PU film (42.2%) and foam (13.94%). The combined system also showed a 1.80 time increase in the production of the monomer MDA, and a 1.23 and 3.62 times increase in the production of AA and BDO, respectively, compared to their production recorded after treatment with only GatA250 or LCC. This study provides valuable insights into PU pollution control and also proposes applicable solutions to manage PU wastes through bio-recycling.

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.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.