Biochemistry Biochemistry最新文献

Glutamine synthetase (GS) is a ubiquitous enzyme central to nitrogen metabolism, catalyzing the ATP-dependent formation of glutamine from glutamate and ammonia. Positioned at the intersection of nitrogen metabolism with carbon metabolism, the activity of GS is subject to sophisticated regulation. While the intricate regulatory pathways that govern Escherichia coli GS were established long ago, recent work has demonstrated that homologues are controlled by multiple distinct regulatory patterns, such as the metabolite induced oligomeric state formation in archaeal GS by 2-oxoglutarate. Such work was enabled in large part by advances in cryo-electron microscopy (cryoEM) that allowed greater structural access to this large enzyme complex, such as assessment of the large heterogeneous oligomeric states of GS and protein-interactor-GS complexes. This perspective highlights recent advances in understanding GS regulation, focusing on the dynamic interplay between its oligomeric state, metabolite binding, and protein interactors. These interactions modulate GS activity, influencing cellular processes such as nitrogen assimilation, carbon metabolism, and stress responses. Furthermore, we explore the emerging concept of GS “moonlighting” functions, revealing its roles in palmitoylation, cell cycle regulation, and ion channel modulation. These diverse functions highlight a newfound versatility of GS beyond its primary catalytic role and suggest complex roles in health and disease that warrant further study.

The amino-terminal domain of collagen α1(XI) plays a key role in controlling fibrillogenesis. However, the specific mechanisms through which various isoforms of collagen α1(XI) regulate this process are not fully understood. We measured the kinetics of collagen type I self-assembly in the presence of specific collagen α1(XI) isoforms. Molecular dynamics simulations, protein–protein docking studies, and molecular mechanics Poisson–Boltzmann surface area were utilized to understand the molecular mechanisms. In vitro, in silico, and thermodynamic studies demonstrated an isoform-specific effect on self-assembly kinetics. Our results indicate isoform-specific differences in the rate constants, activation energy, and free energy of binding. These differences may result from isoform-specific interaction dynamics and modulation of steric hindrance due to the chemically distinct variable regions. We show that isoform A interacts with collagen type I due in part to the acidic variable region, increasing the activation energy of fibril growth while decreasing the rate constant during the growth phase. In contrast, the basic variable region of isoform B may result in less steric hindrance than isoform A. Isoform 0 demonstrated the highest activation energy and the lowest rate constant during the growth phase. Although the presence of isoforms reduced the rate constants for fibril growth, an increase in total turbidity during the plateau phase was observed compared to controls. Overall, these results are consistent with collagen α1(XI) NTD isoforms facilitating fibrillogenesis by increasing the final yield by reducing the rate of the lag and/or growth phases, while extending the duration of the growth phase.

Traumatic brain injury (TBI) is a serious health condition that affects an increasing number of people, especially veterans and athletes. TBI causes serious consequences because of its long-lasting impact on the brain and its alarming frequency of occurrence. Although the brain has some natural protective mechanisms, the processes that trigger them are poorly understood. Fibroblast growth factor (FGF) proteins interact with receptor proteins to protect cells. Gaps in the literature include how basic-FGF (bFGF) is activated by heparin, can heparin-like molecules induce neural protection, and the effect of allosteric binding on bFGF activity. To fill the gap in our understanding, we applied temperature replica exchange to study the influence of heparin binding to bFGF and how mutations in bFGF influence stability. A new favorable binding site was identified by comparing free energies computed from the potential of mean force (PMF). Although the varied sugars studied resulted in different interactions with bFGF compared to heparin, they each produced structural effects similar to those of bFGF that likely facilitate receptor binding and signaling. Our results also demonstrate how point mutations can trigger the same conformational change that is believed to promote favorable interactions with the receptor. A deeper atomic-level understanding of how chemicals are released during TBI is needed to improve the development of new treatments for TBI and could contribute to a better understanding of other diseases.

Farnesyl pyrophosphate derivatives bearing an additional oxygen atom at position 5 proved to be very suitable for expanding the substrate promiscuity of sesquiterpene synthases (STSs) and the formation of new oxygenated terpenoids. Insertion of an oxygen atom in position 9, however, caused larger restraints that led to restricted acceptance by STSs. In order to reduce some of the proposed restrictions, two FPP-ether derivatives with altered substitution pattern around the terminal olefinic double bond were designed. These showed improved promiscuity toward different STSs. Four new cyclized terpenoids with an embedded ether group were isolated and characterized. In the case of two cyclic enol ethers, also the corresponding "hydrolysis" products, linear hydroxyaldehydes, were isolated. Interestingly, all cyclization products originate from an initial 1 → 12 cyclization unprecedented when native farnesyl pyrophosphate serves as a substrate. We found that the most suitable FPP derivative with an additional oxygen at position 9 does not carry any methyl group on the terminal alkene, which likely reduces steric congestion when the preferred conformation for cyclization is adopted in the active site.

Antibiotics are essential medicines threatened by the emergence of resistance in all relevant bacterial pathogens. The engagement of the molecular targets of antibiotics offers multiple opportunities for resistance to emerge. Successful target engagement often requires passage of the antibiotic from outside into the cell interior through one or two distinct membrane barriers. Resistance can occur by preventing the accumulation of antibiotics in sufficient quantities outside the cell, decreasing the rates of entry into the cell, and modifying the antibiotic or the target once inside the cell. These competing equilibria and rates are the lens through which the balance of antibiotic efficacy or failure can be viewed. The two faces of antibiotics, cell growth inhibition or resistance, are reminiscent of Janus, the Roman god of doorways and beginnings and endings, and offer a framework through which antibiotic discovery, use, and the emergence of resistance can be rationally viewed.

Streptomyces griseolus CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs. We successfully crystallized complexes of wild-type CYP105A1 (WT) and the R84A variant with diclofenac (DIF) or flufenamic acid (FLF). In the WT, the carboxyl group of DIF formed a charged hydrogen bond with Arg84. In contrast, in R84A, the carboxyl group formed two bidentate charged hydrogen bonds with Arg73. The C4' atom of the benzene ring of DIF, which undergoes hydroxylation by WT and R84A, was positioned approximately 4 Å from the heme iron. Binding of FLF was nearly the same in both WT and R84A. The carboxyl group of FLF formed charged hydrogen bonds with Arg73. In both WT and R84A, FLF appeared to be fixed by this charged hydrogen bonding with Arg73 during the reaction, and the C4' atom, which undergoes hydroxylation, must face the heme iron. Thus, the dihedral angles of the two N-C bonds connecting the two benzene rings of FLF needed to rotate by 78° and -71°, respectively. The temperature factors of the F-G loop, helix F, and helix G of R84A were remarkably higher than those of WT. This suggests that these regions in R84A are much more flexible compared to those of WT, which may consequently affect substrate binding and product release.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) is a unique thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the formation of DXP, a branchpoint metabolite required for the biosynthesis of vitamins and isoprenoids in bacterial pathogens. DXPS has relaxed substrate specificity and utilizes a gated mechanism, equipping DXPS to sense and respond to diverse substrates. We speculate that pathogens utilize this distinct gated mechanism in different ways to support metabolic adaptation during infection. DXPS is susceptible to time-dependent inhibition by bisubstrate analogs. We suggest that potential differences in the ligand-gated mechanism that may accompany alternative activities of DXPS homologues may enable the development of species-specific bisubstrate analog inhibitors. Here, we evaluate known bisubstrate analog inhibitors of Escherichia coli DXPS (EcDXPS) against DXPS from Pseudomonas aeruginosa (PaDXPS), a Gram-negative pathogen with a remarkable capacity to adapt to diverse environments. Our results indicate that these inhibitors are significantly less potent against PaDXPS compared to EcDXPS. Acceptor site residues that stabilize the phosphonolactyl-ThDP adduct (PLThDP) of bisubstrate analog d-PheTrAP on EcDXPS are not as critical for stabilization of this PLThDP adduct on PaDXPS. Substitution of EcR99 or the analogous PaR106 reduces the potency of both d-PheTrAP and the simpler BAP scaffold, suggesting a common role of these arginine residues in stabilizing PLThDP adducts. However, although EcR99 is required for potent, time-dependent inhibition of EcDXPS by d-PheTrAP, PaR106 does not appear to govern slow-onset inhibition. This work demonstrates that species-specific targeting of DXPS by bisubstrate analogs is possible and highlights mechanistic differences that should be considered in the design of homologue-specific inhibitors, toward narrow-spectrum approaches targeting DXPS.

Aggregation of α-synuclein (α-Syn) and Lewy body (LB) formation are the key pathological events implicated in Parkinson's disease (PD) that spread in a prion-like manner. However, biophysical and structural characteristics of toxic α-Syn species and molecular events that drive early events in the propagation of α-Syn amyloids in a prion-like manner remain elusive. We used a neuronal cell model to demonstrate the size-dependent native biological activities of α-Syn fibril seeds. Biophysical characterization of the fibril seeds generated by controlled fragmentation indicated that increased fragmentation leads to a reduction in fibril size, correlating directly with the extent of fragmentation events. Although the size-based complexity of amyloid fibrils modulates their biological activities and fibril amplification pathways, it remains unclear how the variability of fibril seed size dictates its specific uptake mechanism into the cells. The present study elucidates the mechanism of α-Syn fibril internalization and how it is regulated by the size of fibril seeds. Further, we demonstrate that size-dependent endocytic pathways (dynamin-dependent clathrin/caveolin-mediated) are more prominent for the differential uptake of short fibril seeds compared to their longer counterparts. This size-dependent preference might contribute to the enhanced uptake and transcellular propagation of short α-Syn fibril seeds in a prion-like manner. Overall, the present study suggests that the physical dimension of α-Syn amyloid fibril seeds significantly influences their cellular uptake and pathological responses in the initiation and progression of PD.

In the wake of the pandemic, peptidyl protease inhibitors with Pro-based rigid Leu mimetics at the P₂ position have emerged as potent drug candidates against the SARS-CoV-2 main protease. This success is intuitively attributed to the enhanced hydrophobic interactions and rigidity of Pro-based rigid Leu mimetics in the literature. However, the tertiary amide of proline P₂ derivatives, which hinders the formation of a critical hydrogen bond with the enzyme active site, and the constrained PP_II conformation, which contradicts the protease preferred β-strand conformation, represent two overlooked disadvantages associated with these inhibitors over traditional inhibitors and, theoretically, should adversely affect their potency. Interestingly, despite these major disadvantages, they maintain or display improved potency compared to traditional peptidyl protease inhibitors. In this study, we uncover a previously unnoticed preference for P₂ residues of the protease inhibitors to adopt the PP_II conformation, regardless of residue identity, in the main protease-bound form of key RNA viruses, deviating from the traditional β-strand conformation. We also demonstrate that Pro-based rigid Leu mimetics at P₂ enhance binding affinity by favoring the enzyme-preferred PP_II conformation and significantly reducing configurational entropy loss upon binding, comparable to that of a typical hydrogen bond. This work also highlights the importance of a multidisciplinary approach to enhance the understanding of structure-activity relationships beyond traditional medicinal chemistry intuition. We believe these findings provide new, deep insights and address a major knowledge gap in the area of peptidyl protease inhibitor design, identifying key drivers behind the success of Pro-based peptidyl protease inhibitors beyond mere rigidity and hydrophobicity.

Janustatin A is a potently cytotoxic polyketide alkaloid produced at trace amounts by the marine bacterial plant symbiont Gynuella sunshinyii. Its biosynthetic terminus features an unusual pyridine-containing bicyclic system of unclear origin, in which polyketide and amino acid extension units appear reversed compared to the order of enzymatic modules in the polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line. To elucidate unknown steps in heterocycle formation, we first established robust genome engineering tools in G. sunshinyii. A combination of gene deletion, complementation, production improvement, and NMR experiments then demonstrated that two desaturase homologues, JanA and JanB, are involved in hydroxylation and pyridine formation by desaturation, respectively. Structure-activity relationship studies showed that these modifications substantially increase the cytotoxicity and that the fully functionalized heterocyclic system is crucial for sub-nanomolar cytotoxicity. Isolation of the early post-PKS intermediate janustatin D with an already reversed heterocycle topology supports a noncanonical rearrangement process occurring on the PKS-NRPS assembly line.