Site-specific Mutations Research Articles

Malonylome analysis uncovers the association of lysine malonylation with metabolism and acidic stress in pathogenic Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the pathogenic agent of tuberculosis, remains a primary inducement of morbidity and mortality globally. Mtb have evolved mechanisms to recognize diverse signals, such as acidic pH within phagolysosomes and therefore to reprogram multiple physiological and metabolic processes to adapt to intracellular survival. Moreover, lysine malonylation has been suggested to participate in regulation of enzymes in carbon metabolism. However, lysine malonylation in Mtb and its association with acidic pH associated metabolism adaptation remain unknown. Here, we systematically characterized the comparative malonylome of Mtb H37Rv grown in normal (7H9-Tyloxapol (Ty)-7.4) and acidic (7H9-Ty-4.5) medium mimicking lysosome pH. In total, 2467 lysine malonylation sites within 1026 proteins were identified, which related to diverse biological processes, particularly accumulated in metabolic process. 1090 lysine malonylation sites from 562 proteins were quantified, among which 391 lysine malonylation sites in 273 protein were down-regulated while 40 lysine malonylation sites from 36 proteins were up-regulated in acidic medium, indicating that malonylation may participate in acidic pH associated metabolism. Accordingly, the enzyme activity of GlcB was reduced under acidic stress corresponding to decreased malonylation of GlcB compared with that of normal condition and this was further demonstrated by site-specific mutations. We further found that Mtb-CobB, a sirtuin-like deacetylase and desuccinylase, involved in demalonylase activity. Together, the Mtb malonylome not only indicates the critical role of malonylation in metabolism regulation, but may provide new insights of malonylation on metabolism adaptation to acidic micro-environment in vivo.

Preparation and Validation of Ultra-sensitive Aptamers by Computer Simulation and Site-Specific Mutation

Aptamer is widely used in the detection field as a new type of probe due to their advantages of easy synthesis and modification. Due to the traditional aptamer screening method, the aptamers that are screened by SELEX technology generally have redundant sequences, which affect the binding affinity and detection sensitivity. In this study, a structural transformation strategy for rationally shortening was established by analyzing the secondary structure, finding the sequence of the binding site by base mutation, and double cloning the binding sites. We obtained a 27-mer new aptamer SEQ.A52 with the dissociation constants (Kd) of 10.74 nM, which is 21 times higher than the original aptamer. When used to detect enrofloxacin (ENR) with the fluorescent method, the sensitivity could reach 50 to approximately 1000 pM, and the limit of detections (LOD) was only 41.35 pM, which was 88 times higher than the original aptamer. All the results implied that the strategy could be used for structural modification of some other aptamers to increase affinity and sensitivity.

SQSTM1/p62 promotes miR-198 loading into extracellular vesicles and its autophagy-related secretion

MicroRNA dysregulation is a hallmark of hepatocellular carcinoma (HCC), leading to tumor growth and metastasis. Previous screening on patient specimens identified miR-198 as the most downregulated miRNA in HCC. Here, we show that miR-198 compensation leads to self-release into extracellular vesicles (EVs). Importantly, the vesicular secretion is mediated by autophagy-related pathway, initiated by sequestration of p62/miR-198 complexes in autophagosome-associated vesicle fractions. miR-198 is selectively recognized and loaded by p62 into autophagosomal fractions, whereas mutated miR-198 forms neither induce autophagy and nor interact with p62. Gain and loss of function experiments, using a CRIPR/Cas knockout (KO) and transgenic site-specific p62 mutants, identified p62 as an essential repressor of cellular miR-198 abundancy. Notably, EVs, harboring miR-198/p62 protein complexes, can be uptaken by cells in the close vicinity, leading to change of gene expression in recipient cells. In conclusion, miR-198 enhances autophagy; conversely autophagic protein p62 reduces the miR-198 levels by sorting into extracellular space.Graphical abstractmiR-198 is at first transcribed as primary miRNA, after being processed into single stranded mature miR-198 form, it is transported into cytoplasm ①. By interaction with p62 protein, miR-198 conglomerates and forms a binding complex ②. Since LC3 protein is an interaction partner of p62 protein, hence miR-198 is included into autophagosomes ③. By fusion with multivesicular bodies (MVB), miR-198-binding complex was recruited into amphisomes ④, the latter of which quickly turns into secretory MVB containing intraluminal vesicles⑤. By fusion with cell membrane, intraluminal vesicles were released into extracellular space as EVs ⑥.

Vitamin D and Diseases of Mineral Homeostasis: A Cyp24a1 R396W Humanized Preclinical Model of Infantile Hypercalcemia Type 1.

Infantile hypercalcemia type 1 (HCINF1), previously known as idiopathic infantile hypercalcemia, is caused by mutations in the 25-hydroxyvitamin D 24-hydroxylase gene, CYP24A1. The R396W loss-of-function mutation in CYP24A1 is the second most frequent mutated allele observed in affected HCINF1 patients. We have introduced the site-specific R396W mutation within the murine Cyp24a1 gene in knock-in mice to generate a humanized model of HCINF1. On the C57Bl6 inbred background, homozygous mutant mice exhibited high perinatal lethality with 17% survival past weaning. This was corrected by crossbreeding to the CD1 outbred background. Mutant animals had hypercalcemia in the first week of life, developed nephrolithiasis, and had a very high 25(OH)D3 to 24,25(OH)2D3 ratio which is a diagnostic hallmark of the HCINF1 condition. Expression of the mutant Cyp24a1 allele was highly elevated while Cyp27b1 expression was abrogated. Impaired bone fracture healing was detected in CD1-R396w/w mutant animals. The augmented lethality of the C57Bl6-R396W strain suggests an influence of distinct genetic backgrounds. Our data point to the utility of unique knock-in mice to probe the physiological ramifications of CYP24A1 variants in isolation from other biological and environmental factors.

Enzyme-Catalyzed Spiroacetal Formation in Polyketide Antibiotic Biosynthesis.

A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC. The deletion of olmO abolished oligomycin production and led to the isolation of oligomycin-like metabolites lacking the spiroacetal structure. Purified OlmO catalyzed complete conversion of the major metabolite into oligomycin C. Crystal structures of OssO and OlmO reveal an unusual 10-strand β-barrel. Three conserved polar residues are clustered together in the β-barrel cavity, and site-specific mutation of any of these residues either abolished or substantially diminished OlmO activity, supporting a role for general acid/general base catalysis in spiroacetal formation.

BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer

BackgroundBrother of regulator of imprinted sites (BORIS) is expressed in most cancers and often associated with short survival and poor prognosis in patients. BORIS inhibits apoptosis and promotes proliferation of cancer cells. However, its mechanism of action has not been elucidated, and there is no known inhibitor of BORIS. MethodsA phage display library was used to find the BORIS inhibitory peptides and BTApep-TAT was identified. The RNA sequencing profile of BTApep-TAT-treated H1299 cells was compared with that of BORIS-knockdown cells. Antitumor activity of BTApep-TAT was evaluated in a non-small cell lung cancer (NSCLC) xenograft mouse model. BTApep-TAT was also used to investigate the post-translational modification (PTM) of BORIS and the role of BORIS in DNA damage repair. Site-directed mutants of BORIS were constructed and used for investigating PTM and the function of BORIS.ResultsBTApep-TAT induced DNA damage in cancer cells and suppressed NSCLC xenograft tumor progression. Investigation of the mechanism of action of BTApep-TAT demonstrated that BORIS underwent ADP ribosylation upon double- or single-strand DNA damage. Substitution of five conserved glutamic acid (E) residues with alanine residues (A) between amino acids (AAs) 198 and 228 of BORIS reduced its ADP ribosylation. Inhibition of ADP ribosylation of BORIS by a site-specific mutation or by BTApep-TAT treatment blocked its interaction with Ku70 and impaired the function of BORIS in DNA damage repair.ConclusionsThe present study identified an inhibitor of BORIS, highlighted the importance of ADP ribosylation of BORIS, and revealed a novel function of BORIS in DNA damage repair. The present work provides a practical method for the future screening or optimization of drugs targeting BORIS.

Site-specific target-modification mutations exclusively induced by the coexposure to low levels of pesticides and streptomycin caused strong streptomycin resistance in clinically relevant Escherichia coli

Environmentally relevant levels of pesticides may promote the emergence of stronger streptomycin-resistant mutants in an Escherichia coli K-12 strain in the presence of sublethal levels of streptomycin. However, it is not clear whether this synergistic effect exists in other strains within and outside the Escherichia genus. Here, we investigated the long-term evolution toward stronger antibiotic resistance under the pesticides and streptomycin coexposure in bacterial strains of three different genera, including pathogenic E. coli strains O157:H7 and O103:H2, one Pseudomonas strain, and one Staphylococcus strain. Consistently, the coexposure induced significantly stronger streptomycin-resistant mutants in the two E. coli strains. However, it did not promote any evolution toward stronger streptomycin resistance in the Pseudomonas and Staphylococcus strains. Site-specific mutations of genes, such as rpsL(Lys88Arg), which encode streptomycin target proteins were exclusively evolved in the coexposed E. coli strains and conferred 80-fold increase in streptomycin resistance. These findings imply that a higher risk of strong and inheritable streptomycin resistance of E. coli strains, including the pathogenic ones, may exist in certain environments where pesticides and antibiotics cooccur.

Patients deriving long-term benefit from immune checkpoint inhibitors demonstrate conserved patterns of site-specific mutations

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy and are now the preferred treatment for several tumor types. Though ICIs have shown remarkable efficacy in several cancer histologies, in many cases providing long-term disease control, not all patients will derive clinical benefit from such approaches. Given the lack of a reliable predictive biomarker for therapeutic responses to ICIs, we conducted a retrospective analysis of publicly available genomic data from a large pan-cancer cohort of patients receiving ICI-based immunotherapy. Consistent with previous results, patients in the combined cohort deriving a long-term survival benefit from ICIs were more likely to have a higher tumor mutational burden (TMB). However, this was not uniform across tumor-types, failing to predict for long-term survivorship in most non-melanoma cancers. Interestingly, long-term survivors in most cancers had conserved patterns of mutations affecting several genes. In melanoma, this included mutations affecting TET1 or PTPRD. In patients with colorectal cancer, mutations affecting TET1, RNF43, NCOA3, LATS1, NOTCH3, or CREBBP were also associated with improved prognosis, as were mutations affecting PTPRD, EPHA7, NTRK3, or ZFHX3 in non-small cell lung cancer, RNF43, LATS1, or CREBBP mutations in bladder cancer, and VHL mutations in renal cell carcinoma patients. Thus, this study identified several genes that may have utility as predictive biomarkers for therapeutic responses in patients receiving ICIs. As many have no known relationship to immunotherapy or ICIs, these genes warrant continued exploration, particularly for cancers in which established biomarkers such as PD-L1 expression or TMB have little predictive value.

Ethylene insensitive 2 (EIN2) as a potential target gene to enhance Fusarium head blight disease resistance

Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) severely affects cereal crops, especially wheat and barley. FHB results in significant yield loss, reduces grain quality and contaminates grains with mycotoxin. The development of FHB-resistant cereal cultivars can be expedited through CRISPR gene editing. The Arabidopsis ethylene insensitive 2 (AtEIN2) plays a key role in ethylene signaling pathway and is critical for monitoring plant growth and defense responses. RNAi down-regulation of the wheat homolog TaEIN2 has been shown to enhance wheat FHB resistance. Here we generated site-specific mutations in AtEIN2 by CRISPR-editing. Detached inflorescence infection assays revealed that AtEIN2 knock-out (KO) mutants displayed enhanced Fg resistance and substantially reduced Fg spore production in planta. Gene expression profiling of defense genes revealed that impairment of AtEIN2 resulted in down-regulation of the ethylene signaling pathway while the salicylic acid signaling pathway was unaffected. Complementation of AtEIN2-KO plants with a barley orthologue, HvEIN2, restored Fg susceptibility, indicating that HvEIN2 is functionally equivalent to its Arabidopsis counterpart and, hence, may have a similar role in conditioning barley Fg susceptibility. These results provide insight into the defense role of EIN2 and a molecular and functional foundation for manipulating HvEIN2 to enhance FHB resistance in barley.

Selectively Fluorinated Citronellol Analogues Support a Hydrogen Bonding Donor Interaction with the Human OR1A1 Olfactory Receptor.

C-2 fluorinated and methylated stereoisomers of the fragrance citronellol 1 and its oxalate esters were prepared from (R)-pulegone 11 and explored as agonists of the human olfactory receptor OR1A1 and assayed also against site-specific mutants. There were clear isomer preferences and C-2 difluorination as in 18 led to the most active compound suggesting an important hydrogen bond donor role for citronellol 1. C-2 methylation and the corresponding oxalate ester analogues were less active.

Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing.

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10E9-recognized 84SAGKDP89 epitope and 3F3-recognized 320ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Novel Functional FOXC1 Variants in Familial and Sporadic Atrial Septal Defect

ObjectiveAtrial septal defect (ASD) is one of the most common forms of congenital heart disease. Genetic defects play important roles in the pathogenesis of ASD but are complex and unclear. This study tries to discover the genetic pathogenesis in familial and sporadic ASD patients.MethodsIn Stage I, in a family with 30 members whole exome sequencing was performed in 4 ASD patients and the identified genetic variants were screened in the rest of the family members. In Stage II, 335 unrelated sporadic and isolated ASD patients were enrolled to test whether the genetic variants were involved in these patients. In Stage III, biological functions of the variants were elucidated at the cellular level and the CRISPR/Cas9 was used to construct variant‐specific mutant mice in order to observe the abnormality of the heart structure.ResultsA novel heterozygous missense variant, FOXC1 c.518G>A (p.R173H) was identified in the ASD family by whole exome sequencing. Subsequently, other 4 heterozygous variants in FOXC1, including 2 novel missense variants: c.556‐558delAAG (p.K186del) and c.559G>A (p.D187N) were identified in unrelated 335 sporadic ASD patients. In dual‐luciferase reporter assay, the transcriptional activity of R173H decreased to almost 30% of the wild‐type. In the Foxc1 R173H site‐specific mutant mice, the atrial septum became very thin in tissue slices of the heart that was similar to the echocardiographic finding in the above family members.ConclusionVariants in FOXC1 likely cause cardiac anomaly, particularly ASD. These findings provide a new insight into genetic mechanisms and counseling of familial and sporadic ASD.

Cysteine-Based Mimic of Arginylation Reproduces Neuroprotective Effects of the Authentic Post-Translational Modification on α-Synuclein.

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.

Alkaloid Biosynthetic Enzyme Generates Diastereomeric Pair via Two Distinct Mechanisms.

Ergopeptines constitute one of the representative classes of ergoline alkaloids and carry a tripeptide extension on the lysergic acid core. In the current study, we discovered and structurally characterized newly isolated ergopeptine-like compounds named lentopeptins from a filamentous fungus Aspergillus lentulus, a close relative of A. fumigatus. Interestingly, in lentopeptins, the common lysergic acid moiety of ergopeptines is replaced by a cinnamic acid moiety at the N-terminus of the peptide segment. Moreover, lentopeptins lack the C-terminal proline residue necessary for the spontaneous cyclization of the peptide extension. Herein, we report the atypical lentopeptin biosynthetic pathway identified through targeted deletion of the len cluster biosynthetic genes predicted from the genome sequence. Further in vitro characterizations of the thiolation-terminal condensation-like (T-CT) didomain of the nonribosomal peptide synthetase LenA and its site-specific mutants revealed the mechanism of peptide release via diketopiperazine formation, an activity previously unreported for CT domains. Most intriguingly, in vitro assays of the cytochrome P450 LenC illuminated the unique mechanisms to generate two diastereomeric products. Lentopeptin A forms via a stereospecific hydroxylation, followed by a spontaneous bicyclic lactam core formation, while lentopeptin B is produced through an initial dehydrogenation, followed by a bicyclic lactam core formation and stereospecific hydration. Our results showcase how nature exploits common biosynthetic enzymes to forge new complex natural products effectively (213/250).

Occidiofungin Is the Key Metabolite for Antifungal Activity of the Endophytic Bacterium Burkholderia sp. MS455 Against Aspergillus flavus.

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Slow Conformational Changes of Blue Light Sensor BLUF Proteins in Milliseconds.

Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization

Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size.

Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ54-dependent, butanol-responsive TF–promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Cellular synthesis of protein pretzelanes

Topology has been recognized as a unique dimension in molecular engineering, yet the topological diversity remains largely untapped, especially in macromolecules. Herein, we report the molecular design, cellular synthesis, and detailed characterization of protein pretzelanes with a chemical topology of a bridged Hopf link. The synergy between the intramolecular chain entwining guided by the p53dim (X) domains and the genetically encoded side-chain coupling by SpyTag(A)-SpyCatcher(B) reaction facilitates the direct synthesis of the model protein pretzelane BXA-BXA in Escherichia coli. The approach tolerates the insertion of various proteins-of-interest, such as elastin-like protein (ELP), superfolder green fluorescent protein (GFP) and dihydrofolate reductase (DHFR), at the bridge region between two rings, giving rise to three protein pretzelanes BXA-ELP-BXA, BXA-GFP-BXA, and BXA-DHFR-BXA. Their topology has been verified by combined techniques of MALDI-TOF mass spectrometry, ion mobility-mass spectrometry, site-specific mutation, and orthogonal proteolytic digestion experiments. Not only are the fluorescent properties of GFP and the catalytic properties of DHFR fully retained, the pretzelane topology also renders BXA-DHFR-BXA more thermally resilient than the wild-type DHFR. These results expand the topological diversity of proteins and demonstrate protein stabilization as a potential functional benefit for the pretzelane topology.