Substrate Affinity Research Articles

NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.

Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4's redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF's cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.

Uricase from Alkalihalobacillus clausii, a Candidate for Industrial Application of Reducing Uric Acid Content of Bean Products.

Microbial uricase is an essential enzyme in purine degradation and the development of low-purine food. High enzyme activity and an appropriate optimum pH must be established for low-purine food. Uricases from Neurospora crassa, Streptomyces coelicolor, Alkalihalobacillus clausii, Bacillus subtilis, and Brevibacterium casei were heterologously expressed in Escherichia coli. Uricase from Alkalihalobacillus clausii (AC-PUCL) showed the most potent enzyme activity (249.19 IU/mL) at 37 °C and pH 7.0. This is close to the pH of plant-based food. The Km and Km/Kcat values of AC-PUCL were 30.12 μM and 1.46 s-1 μM-1, respectively. Furthermore, the crystal structures of uricases from different sources revealed that hydrogen bonds could enhance substrate affinity and strong enzyme activity. In addition, the high enzyme activity may be contributed by the active pockets with an appropriate size. Finally, AC-PUCL helped reduce the purine substances in soybean, pea, and kidney bean, with residual uric acid can not be detected at pH 8.6, suggesting a promising industrial application.

Overexpression of ace2 compensating for the acetylcholinesterase activity loss from ace1 mutations accelerated the development of eggs and early nymphs in Nilaparvata lugens.

Acetylcholinesterase (AChE) guarantees the acetylcholine signal in insect central nervous system, and is the target of organophosphorus and carbamate insecticides, towards which resistance was often reported due to AChE mutations. In Nilaparvata lugens, a major pest on rice, two mutations (G119S and F331C) were detected in AChE1 in a chlorpyrifos-resistant (CHL) strain. The double mutations in AChE1 reduced total AChE activity in metabolizing acetylcholine, such as low protein stability, substrate affinity and catalytic efficiency, which needed compensation in a special way. In CHL strain, the transcriptional level of ace2 encoding AChE2 was systematically elevated, such as over 30-fold overexpression in brain. The ace2 overexpression not only compensated for AChE activity loss in brain due to AChE1 mutations, but also accelerated the development of eggs and early nymphs in CHL strain. When performing ace2 RNAi in CHL eggs, the egg and early nymph duration were recovered. In CHL eggs, the transcriptional levels of three basic helix-loop-helix transcription factors (Ase2, Ato1 and SCL), which were closely related to neural development, were significantly upregulated. Their respective RNAi in CHL strain also significantly recovered the egg duration, as RNAi towards ace2, which partially explained the reason for the accelerated development of eggs and early nymphs. The results revealed AChE2 non-canonical function in insect embryonic development, and uncovered a physiological effect caused by ace2 overexpression as a compensation action for resistance mechanism due to AChE1 mutations, providing a base for insecticide resistance management in insects.

Crystal structures of Cystathionine β-lyase and Cystathionine β-lyase like protein from Bacillus cereus ATCC 14579

Cystathionine β-lyase (CBL) and cystathionine β-lyase-like protein (CBLP) are key PLP-dependent enzymes involved in methionine biosynthesis. In Bacillus cereus ATCC 14579 CBL (BcCBL) and CBLP (BcCBLP) catalyze the conversion of cystathionine to homocysteine and pyruvate. In this study, we found that both BcCBL and BcCBLP effectively catalyze cystathionine cleavage, with BcCBLP exhibiting a higher catalytic efficiency (kcat) and low substrate affinity (Km). We determined their crystal structures in complex with pyridoxal phosphate (PLP). BcCBL, forming a tetramer, aligns with typical CBLs in sulfur amino acid metabolism, while BcCBLP, forming a dimer, resembles the bifunctional MalY enzyme from Escherichia coli, indicating potential additional regulatory roles. These structural and functional insights highlight the distinct roles of BcCBL and BcCBLP in cellular metabolism. This study provides valuable insights into the structural diversity and potential functions of these enzymes, contributing to the broader knowledge of PLP-dependent enzymatic mechanisms.

Single-atom nanozyme-based catalytic ROS clearance for efficiently alleviating eczema.

Single-atom nanozymes (SAzymes) with excellent biological catalytic activity have emerged as promising candidates for advancing biomedical applications. Herein, we synthesized a RuN4-SAzyme by thermal decomposition. In in vitro experiments, the RuN4-SAzyme demonstrated exceptional catalytic efficiency in mimicking the activity of peroxidase, with a Michaelis-Menten constant (Km) for 3,3',5,5'-tetramethylbenzidine reaching 0.077 mM, reflecting a high substrate affinity. Moreover, the RuN4-SAzyme also exhibited catalase- and superoxide dismutase-like activities, as well as nicotinamide adenine dinucleotide oxidase-like activity, effectively neutralizing reactive oxygen species. Significantly, in vivo experiments demonstrated that RuN4-SAzymes can effectively alleviate eczema-like symptoms by clearing inflammatory factors, reducing oxidative stress, and promoting macrophage polarization from M1 to M2, showing comparable efficacy to the clinical drug tacrolimus.

Lysine 204 is crucial for the antiport function of the human LAT1 transporter

LAT1 (SLC7A5) catalyzes an antiport reaction of amino acids with specificity towards the essential ones. It is mainly expressed at the Blood Brain Barrier and placenta barriers, but it becomes over-expressed in virtually all human cancers even if originating from tissues with lower expression levels. The antiport reaction of LAT1 is crucial at the BBB since its inherited loss causes Autism Spectrum Disorder. We have investigated the possible molecular determinant of the antiport by site-directed mutagenesis, in vitro transport assay and computational analysis. Previous data indicated that mutation of K204 impairs, but does not knock out LAT1 functionality. We have investigated the activity changes in the K204Q mutant by following the transport of [3H]-histidine, one of the major substrates, in proteoliposomes harbouring the WT or K204Q. In the mutant, the [3H]-histidine uptake and efflux are not more stimulated by the counter-substrate as they occur in the WT. Moreover, the mutation strongly decreases the substrate affinity and alters the pH dependence of K204Q. Molecular Dynamics analysis correlates well with the experimental data since it shows that substrate prematurely escapes the binding site. In addition, the K204Q shows a strongly increased mobility in those regions, transmembrane domains and random coils, involved in the transport cycle. The identified Lys residue could be responsible of the same phenomenon in those members of the SLC7 family, described as antiporters, in which it is conserved.

Vulcanization Accelerators and Silica Coupling Agents in Polyisoprene Melts.

The achievement of sufficient dispersion of vulcanization accelerators is critical to tailoring superior cross-linked elastomers. Modern recipes rely on multicomponent formulations with silica particles covered by coupling agents. We study the molecular properties of select accelerators in polyisoprene melts and their affinity for functionalized surfaces via extensive all-atom molecular dynamics simulations. We focus on the common (N-cyclohexyl)-2-benzothiazole sulfenamide (CBS), 1,6-bis(N,N-dibenzylthiocarbamoyldithio)hexane (DBTH), and diphenyl guanidine (DPG) molecules and their mixing characteristics at curing temperatures. Our results support a low self-association affinity for CBS and DBTH within polyisoprene, whereas DPG forms small hydrogen-bonded aggregates. Subsequently, we examine systems in contact with silica interfaces, bare or grafted with (3-mercaptopropyl)triethoxysilane (MPTES), (3-octanoylthio) 1-propyl-triethoxysilane (NXT), and bis[3-(triethoxysilyl)propyl]disulfide (TESPD). Accelerator-substrate affinity is first assessed at infinite dilution using free energy calculations and subsequently at finite concentrations. Accelerators exhibit high substrate affinity (DPG > CBS > DBTH) irrespective of functionalization. However, coupling agents are able to displace from the surface a significant amount that increases with the grafting density and the size of the coupling agent. Finally, we investigate the behavior of DPG in binary DPG-CBS formulations, where the former can act as a covering agent that solubilizes CBS into the bulk polymer.

Molecular Structure of Paraoxonase-1 and Its Modifications in Relation to Enzyme Activity and Biological Functions-A Comprehensive Review.

PON1 is a Ca2+-dependent enzyme that indicates a hydrolytic activity towards a broad spectrum of substrates. The mechanism of hydrolysis catalyzed by this enzyme is poorly understood. It was shown that the active site of PON1 is highly dynamic. The catalytic center of this enzyme consists of side chains of amino acids binding two calcium ions, from which the first one performs a structural function and the other one is responsible for the catalytic properties of PON1. This review summarizes available information on the structure of PONs, the role of amino acids located in the active site in specificity, and multiple substrate affinity of enzymes for understanding and explaining the basis of the physiological function of PONs. Moreover, in this paper, we described the changes in the structure of PONs induced by environmental and genetic factors and their association with diseases. The detoxification efficiency depends on the polymorphism of the PON1 gene, especially Q192R. However, data on the association between single-nucleotide polymorphisms (SNPs) in the PON1 gene and cardiovascular or neurodegenerative diseases are insufficient. The reviewed papers may confirm that PON1 is a very promising tool for diagnostics, but further studies are required.

Enhanced Bioactivity of Quercetin-Tetrahydroisoquinoline Derivatives: Effect on Lipophilicity, Enzymes Inhibition, Antioxidant Potential, and Cytotoxicity.

Quercetin, a well-known flavonoid with significant medicinal potential, was derivatized at the C8 position with a tetrahydroisoquinoline (THIQ) moiety, and physicochemical and pharmacological properties, inhibition potential, antioxidant activity, and cytotoxicity of new compounds were evaluated. Physicochemical and pharmacological properties, including lipophilicity, membrane permeability, and P-glycoprotein substrate affinity, were assessed theoretically using the SwissADME software. The metal-chelating ability of the new compounds was evaluated on metal ions Fe2+, Zn2+, and Cu2+, whose homeostasis disruption is linked to the development of Alzheimer's disease. Inhibition potential was tested on the cholinergic enzymes acetylcholinesterase and butyrylcholinesterase, as well as Na+, K+-ATPase, an enzyme commonly overexpressed in tumours. Antioxidant potential was assessed using the DPPH assay. Cytotoxicity studies were conducted on healthy MRC-5 cells and three cancer cell lines: HeLa, MDA-231, and MDA-468. The results indicated that derivatization of quercetin with THIQ yielded compounds with lower toxicity, preserved chelating ability, improved antioxidant potential, increased selectivity toward the cholinergic enzyme butyrylcholinesterase, and enhanced inhibition potential toward Na+, K+-ATPase and butyrylcholinesterase compared to quercetin alone. Therefore, the synthesized derivatives represent compounds with an improved profile and could be promising candidates for further optimization in developing drugs for neurodegenerative and cancer diseases.

Structure of the SMYD2-PARP1 Complex Reveals Both Productive and Allosteric Modes of Peptide Binding.

Allosteric regulation allows proteins to dynamically respond to environmental cues by modulating activity at sites away from the catalytic center. Despite its importance, the SET-domain protein lysine methyltransferase superfamily has been understudied. Here, we present four crystal structures of SMYD2, a unique family member with a MYND domain. Our findings reveal a novel allosteric binding site with high conformational plasticity and promiscuity, capable of binding peptides, proteins, PEG, and small molecules. This site exhibits positive cooperativity with substrate binding, influencing catalytic activity. Mutations here significantly alter substrate affinity, changing the enzyme's kinetic profile. Specificity studies show interaction with PARP1 but not histones, suggesting targeted regulation. Interestingly, this site's function remains unaffected by active site changes, indicating unidirectional mechanisms. Our discovery provides novel insights into SMYD2's biochemical regulation and lays the foundation for broader research on allosteric control in lysine methyltransferases. Given SMYD2's role in various cancers, this work opens exciting avenues for designing specific allosteric inhibitors with reduced off-target effects.

A novel dual-function biomimetic approach for high throughput organic dye biodegradation and hydrogen peroxide sensing using a nanosized artificial peroxidase with ultra-improved substrate affinity and superb catalytic efficiency

A novel dual-function biomimetic approach for high throughput organic dye biodegradation and hydrogen peroxide sensing using a nanosized artificial peroxidase with ultra-improved substrate affinity and superb catalytic efficiency

The discovery of a random endo-acting ι-carrageenase with bifunctional activities against ι-carrageenan and κ-carrageenan.

Carrageenans have attracted increasing research interests in recent decades for their various physicochemical and physiological properties. Random endo-acting carrageenases are promising tools for tailoring the molecular weight of carrageenan and preparing a series of carrageenan oligosaccharides. Although the processive ι-carrageenases in the GH82 family have been widely investigated, the random ι-carrageenase has not been reported. Herein, a novel GH82 family protein Cg82Mf, which was identified by AlphaFold2 as lacking a lid structure on the catalytic groove, was cloned and expressed in Escherichia coli. The analysis of hydrolysis pattern proved that Cg82Mf was the first random endo-acting enzyme against ι-carrageenan, and was capable of preparing the hydrolysis products with various degrees of polymerization. Cg82Mf exhibited higher substrate affinity among all characterized ι-carrageenases, reflected by its low Km value (0.18 μM). Remarkably, Cg82Mf could also hydrolyze κ-carrageenan, that is, the subsites of the enzyme could tolerate κ-carrageenan disaccharide, which demonstrated a novel cleavage specificity. The novel hydrolysis pattern and cleavage specificity shed light on the presence of diversity within the GH82 family, and indicated that Cg82Mf could be facilitated as a potential biocatalyst for the molecule tailoring of carrageenans.

Exploration of urease-aided calcium carbonate mineralization by enzyme analyses of Neobacillus mesonae strain NS-6.

Urease containing nickel cofactor is crucial for urea-hydrolytic induced calcium carbonate (CaCO3) precipitation (UICP). However, limited information exists regarding the influence of amino acid residues interacting with nickel ions in its structure on induced CaCO3 mineralization. Herein, RT-qPCR was used to demonstrate that the addition of NiCl2 dramatically upregulated the expression of urease structural gene ureC that was correlated with nickel binding in Neobacillus mesonae strain NS-6. Homology modeling and molecular docking were employed to construct the three-dimensional structure of urease and seek the key residues involved in nickel binding process, and virtual mutation technology was adopted to inform three key residues coordinated with nickel ions and urea, His249, His275, and Asp363. Four metrics, including root mean square deviation values for mutations of those key residues in urease-urea complexes severally and wild-type, were calculated by molecular dynamics simulations when they were mutated into alanine, respectively. Subsequently, the mutations of H249A, H275A, and D363A were characterized using western blotting to reveal a decrease in the relative expression and activity of urease, along with a corresponding reduction in CaCO3 precipitation. Ultimately, the mutations also exhibited that they had lower substrate affinity and catalytic efficiency for urea through enzymatic properties analysis. The findings suggested that those residues played a pivotal role in UICP of strain NS-6, which would expand the theoretical basis for modulating urease activity.IMPORTANCEUrease-producing bacterium is of great importance in diverse application fields, such as environmental remediation, due to its key driving characteristics in catalyzing urea hydrolysis via urea-hydrolytic induced CaCO3 precipitation (UICP). As essential cofactors of urease, nickel ions play a crucial role in regulating urease catalysis and maintaining structural stability. Numerous investigations have emphasized the impact of nickel ions on urease activity in recent years, to our best knowledge, only a few literatures have studied the molecular-level regulation of nickel-ligand residues. This study focused on the highly urease-producing bacterial Neobacillus mesonae NS-6 to explore the effects of specific nickel-ligand residues on the urease-aided CaCO3 mineralization process using molecular simulation predictions and targeted mutation experiments. The aim was to provide a molecular-level understanding of the interactive effects between urea and critical residues associated with the urease active center, as well as propose an effective modification strategy to enhance the application of UICP in future environmental areas.

Tetramerization of deoxyadenosine kinase meets the demands of a DNA replication substrate challenge in Giardia intestinalis.

The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on salvaging deoxyadenosine and other deoxyribonucleosides from its host environment. Here, we report that G. intestinalis has a deoxyribonucleoside kinase with a 1000-fold higher catalytic efficiency (kcat/KM) for deoxyadenosine than the corresponding mammalian kinases and can thereby provide sufficient deoxyadenosine triphosphatelevels for DNA synthesis despite the lack of de novo synthesis. Several deoxyadenosine analogs were also potent substrates and showed comparable EC50 values on cultured G. intestinalis cells as metronidazole, the current first-line treatment, with the additional advantage of being effective against metronidazole-resistant parasites. Structural analysis using cryo-EM and X-ray crystallography showed that the enzyme is unique within its family of deoxyribonucleoside kinases by forming a tetramer stabilized by extended N- and C-termini in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a markedly reduced affinity for the deoxyribonucleoside substrate. The development of highly efficient deoxyribonucleoside kinases via oligomerization may represent a critical evolutionary adaptation in organisms that rely solely on deoxyribonucleoside salvage.

A simple, rapid, and low-cost approach for colloidal nanoparticle-based surface enhanced Raman Scattering detection of endosulfan pesticide at trace levels

Abstract Surface Enhanced Raman Spectroscopy (SERS) is a highly specific, sensitive, and portable technique with great potential for on-site pesticide detection and monitoring. Endosulfan, an organochlorine pesticide known for its high toxicity, slow degradation, and bioaccumulation, has poor affinity for metallic SERS substrates. This study presents a label-free SERS detection method for endosulfan, using aggregating agents like potassium chloride (KCl), potassium hydroxide (KOH), potassium bromide (KBr), and sodium hydroxide (NaOH) to modify the behavior of Ag colloidal nanoparticles (NPs) and enhance the SERS signal of endosulfan molecules trapped within formed hot-spots. We analyzed the UV–Vis spectra, the hydrodynamic diameter, and zeta-potential of Ag NPs with the addition of these agents and endosulfan. Successful detection of both α- and ß- endosulfan isomers at μM concentrations in both ethanol and methanol was achieved with KOH-treated Ag NPs. The method was also applied to detect endosulfan in real water samples, along with simultaneous detection of λ-cyhalothrin, showcasing its capability to identify multiple analytes. The selectivity and specificity were confirmed using a mixture of endosulfan and thiabendazole, highlighting the crucial role of selecting the appropriate aggregating agent for each analyte. Overall, the findings emphasize the potential of aggregating agents to mediate the SERS enhancement of endosulfan, facilitating simple and rapid protocols for environmental pollutant detection, while shedding light on the intricate interplay between NP behavior, surface chemistry, and analyte interaction.

Immobilization of snailase on glutamate modified MIL-88B(Fe) to efficiently convert the rare ginsenoside CK with high enzyme recyclability and stability

The carboxyl groups on MIL-88B(Fe) are crucial for the covalent immobilization of snailase, and the enzyme can convert common ginsenoside Rb1 into the rare ginsenoside compound K (CK) with higher bioavailability. The present study proposed glutamate-modified MIL-88B(Fe) for the immobilization of snailase to improve enzymatic activity and loading capacity. The surface topography characterized by SEM and CLSM indicated snailase was successfully encapsulated and uniformly distributed in the Sna@MIL-88B(Fe). The maximum immobilized capacities of snailase by MIL-88B(Fe)-Glu and MIL-88B(Fe) were 185 mg/g and 140 mg/g, respectively. Moreover, covalently immobilized snailase on MIL-88B(Fe)-Glu showed better pH, thermal, solvent, and storage stabilities than those immobilized on MIL-88B(Fe) and resolvase. Meanwhile, the reaction kinetics exhibited that the Km value of Sna@MIL-88B(Fe)-Glu (1.6 mM) was significantly lower than that of free snailase (2.1 mM), indicating a higher substrate affinity. Besides, more ginsenoside CK with higher conversion (60.71 %) was generated by Sna@MIL-88B(Fe)-Glu, even after five cycles. The glutamate modified covalent grafting method provides a highly efficient strategy for biocatalysis and a reference for the immobilized snailase-catalyzed transformation of rare ginsenosides CK.

Rational design to enhance the catalytic activity of acetylcholinesterase and mitigate trichlorfon toxicity in vitro

Trichlorfon (TCF) is a widely used organophosphate pesticide whose inhibition of acetylcholinesterase (AChE) results in neurotoxicity and significant biosafety risks. Addressing these concerns requires effective strategies to mitigate TCF-induced toxicity and safeguard exposed organisms. In this study, we explored the potential of a catalytic activity enhanced Culex pipiens AChE mutant to mitigate TCF-induced cytotoxicity through rational design. A double-point mutant, M5 (I198M/Y249F), was developed by combining molecular dynamics (MD) simulations with structural feature analysis to reshape the active pocket, which demonstrated enhanced catalytic efficiency and maintained thermostability. Its functional activity and improved catalytic performance were further confirmed by activity staining on non-denaturing gels. The analysis of the catalytic mechanism and the reduction in Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) free energy revealed an increase in substrate affinity for M5. Additionally, the application of exogenous M5 not only restored endogenous AChE activity in NIH/3T3 cells exposed to TCF but also reduced reactive oxygen species (ROS) accumulation and apoptosis, thereby improving cell viability. In silico studies indicate that the stable interaction between M5 and TCF promotes the targeted depletion of TCF, effectively neutralizing its toxic effects. These findings indicate that M5 has potential as an enzyme-based antidote for organophosphate pesticide, offering a novel strategy for protecting non-target species from pesticide-induced damage.

Engineering the Non-catalytic Domain to Enhance Catalytic Activity and Thermal Stability of a Nκ2-Producing κ-Carrageenase.

κ-Carrageenases play an important role in achieving the high-value utilization of carrageenan polysaccharides. They can be used in the preparation of even-numbered κ-neocarrageenan oligosaccharides by degrading κ-carrageenan (KC). We previously identified and characterized a κ-carrageenase, CaKC16B, with high specificity for producing a single κ-neocarrabiose. It can produce a single κ-neocarrabiose from degrading KC. However, they also exhibited poor thermal stability and catalytic efficiency. To improve these properties, we introduced noncatalytic domains (nonCDs) from a heat-resistant κ-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct CaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of them exhibited improved enzymatic properties, including optimal reaction temperature, thermal stability, substrate affinity, and catalytic efficiency. Remarkably, the kcat/Km values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and βκ-carrageenan, with mutants exhibiting higher relative activity for βκ-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and βκ-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.

The faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis

We propose here that acylation modification of actinomycete proteins is a restrictive system that limits the excessive synthesis of secondary metabolites, its mechanism has not been clearly elucidated before. We used crotonylation as an example to investigate the acylation effect in the daptomycin biosynthesis by Streptomyces roseosporus. Our experiments revealed abundant crotonylation of numerous secondary metabolic enzymes in Streptomyces roseosporus, a daptomycin producer. DptE, which initiates daptomycin biosynthesis, is crotonylated at K454. We experimentally identified the corresponding DptE crotonyltransferase Kct1 and decrotonylase CobB. Further studies consistently confirmed that decrotonylation increases DptE activity. Decrotonylation functions like loosening a faucet knob, increasing substrate channel throughput and the initial flow of daptomycin biosynthesis. Moreover, DptE catalytic activity was enhanced via K454 and neighboring residues K184 and Q420 mutation, increasing daptomycin yield by 132%; daptomycin biosynthesis related metabolism activities also increased. Substrate channel prediction revealed 38% higher throughput for mutant DptE (K454I/K184Q/Q420N) than crotonylated DptE. Molecular dynamics (MD) simulations revealed significant increases in flexibility and substrate affinity of the mutant. In summary, we elucidated the faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis and adopted decrotonylation to generate high-yield industrial strains.

A proteolytic AAA+ machine poised to unfold protein substrates

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP’s ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX’s ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.