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Gamma-glutamyl peptidase 1 (GGP1) plays a dual role in primary and secondary sulfur metabolism in Arabidopsis thaliana. During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into glutamate and cysteinylglycine. During glucosinolate and camalexin biosynthesis, GGP1 processes GSH conjugates by hydrolyzing the same isopeptide bond of γ-glutamate. In the present study, we determined the crystal structures of the following GGP1 forms: ligand-free, glutamate complex, covalent γ-glutamate intermediate, and disulfide-linked S-S inactive forms. The intermediate structure, in which γ-Glu is covalently linked to the catalytic nucleophile cysteine (C100), was trapped by mutating the catalytic histidine to asparagine (H192N). In the glutamate complex and γ-glutamate intermediate structures, glutamate bound to the S1 subsite is extensively recognized by several hydrogen bonds. The substrate recognition of the cysteinylglycine moiety at the S1' and S2' subsites was revealed by predicting the complex structure with a GSH conjugate. Mutational analysis indicated that R206 plays an important role in substrate binding by forming a salt bridge with glycine at the S2' subsite. An open pocket is present beyond the thiol side chain of cysteine in the S1' subsite, which contributed to the dual activity of GGP1 toward GSH and the GSH conjugates. The S-S inactive structure was obtained by soaking GGP1 crystals in cysteinylglycine, and C100 partially formed a disulfide bond with a neighboring C154 residue. The partial inactivation of GGP1 in the presence of a pro-oxidant (cysteinylglycine) has suggested its possible role in oxidative stress regulation in Arabidopsis.

Helicobacter pylori is a bacterium that colonizes the stomach and causes gastric disorders in humans. For successful colonization in the harsh gastric environment, H. pylori employs various homeostatic mechanisms in response to environmental factors, such as protons and copper ions. Copper levels should be maintained below toxicity in the cell while remaining above the threshold required for biological functions. Copper resistance determinant A (CrdA) is a putative copper chaperone protein that contributes to copper homeostasis in H. pylori. To provide insight into CrdA-mediated copper homeostasis, we analyzed the interaction of CrdA with Cu(I) or Cu(II) ions through biochemical and mutational studies and determined the crystal structures of CrdA alone and in complex with a Cu(I) mimic, Ag(I). CrdA exhibited a binding preference for Cu(I) and Ag(I) ions over Cu(II) ions. CrdA forms a Greek key β-barrel structure with a unique protruding methionine-rich motif whose methionine residues coordinate two Ag(I) ions. In particular, the CrdA residue M69 plays a key role in Cu(I) recognition, even at the low pH found in the stomach where H. pylori resides. Furthermore, our structure-based comparative analysis suggests that CrdA has evolved a unique Cu(I) recognition mechanism that has not been observed for other Cu(I) chaperone proteins. Our findings on the specific interaction of CrdA with Cu(I) ions would provide a new avenue for developing H. pylori-targeting antibacterial drugs.

Metabolic pathways are considered to originate from broad-specificity ancestors that later diverged into specialized routes. Thermus thermophilus possesses an unusual amino group carrier protein (AmCP)-mediated lysine biosynthetic pathway alongside a canonical arginine biosynthetic pathway. Although each route is considered specific to its cognate amino acid, several lysine biosynthetic enzymes have been shown to accept arginine intermediates. We herein investigated [LysW]-aminoadipate kinase (LysZ; EC:2.7.2.17) and [LysW]-L-2-aminoadipate 6-phosphate reductase (LysY; EC:1.2.1.103), which catalyze the second and third steps, respectively, in the conversion of α-aminoadipate (AAA) to lysine using amino group carrier protein LysW (AmCP), to define their specificity and evolutionary origin. To examine the potential promiscuity, we engineered LysX variants capable of synthesizing LysW-Glu, an artificial LysW-bound analogue that mimics an arginine pathway intermediate. LysZ exhibited activity for LysW-Glu that was approximately 60% of the original activity for LysW-AAA. The activity of LysY for LysW-Glu phosphate was estimated to be approximately 15-20% of that observed with LysW-AAA phosphate. The present study revealed that both enzymes can also act on an arginine biosynthetic intermediate, but with distinct degrees of efficiency. Phylogenetic reconstructions further suggested that an AmCP-mediated biosynthetic pathway represents a primitive route for the synthesis of lysine and arginine in a primordial cell. More generally, the results obtained herein will contribute to a more detailed understanding of the evolutionary strategies employed by nature to specialize and expand metabolic pathways and adjust enzyme promiscuity.

The biosynthesis of polyprenyl/dolichyl phosphate, an essential lipid carrier in protein glycosylation, occurs across all domains of life. Eukaryotic heteromeric enzymes involved in polyprenyl chain elongation consist of a highly conserved catalytic cis-prenyltransferase subunit (CPT-CS) and a less conserved CPT-accessory subunit (CPT-AS). Here, we present the first experimental evidence that dolichol biosynthesis in Paramecium tetraurelia is mediated by a heteromeric CPT complex. Using a multidisciplinary experimental approach, we identified two highly homologous catalytic CPT subunits, CPT1a and CPT1b, which exhibit high sequence similarity to other eukaryotic CPTs, along with a unique CPT-AS, named POC1 (partner of CPT1), which is a structural and functional relative of the human dehydrodolichyl diphosphate synthase complex subunit NUS1 (also known as NgBR) and yeast Nus1 CPT-AS. Despite low sequence similarity to other CPT-ASs, it retained a well-preserved C-terminal substrate-binding domain characteristic of its eukaryotic and prokaryotic counterparts. The loss of POC1 or CPT1a, but not CPT1b, results in a deficit in dolichol production, leading to a significant reduction in glycoprotein content and, ultimately, to the P. tetraurelia cell death. In a heterologous yeast system, both CPTs in complex with POC1 synthesized polyprenyl chains. The identification of a POC1 protein so distinct from other CPT-ASs may spark further efforts to uncover CPT-AS proteins in pathogenic protozoa, which have so far eluded detection despite phylogenetic evidences that CPT of Apicomplexa and Trichomonas sp. are heteromeric enzymes. Given their substantial sequence divergence from human NgBR and its animal orthologues, these protozoan CPT-ASs could represent highly specific targets for antiparasitic therapies.

Clinical and epidemiological studies suggest similarities in dysregulation of pathways in type 2 diabetes (T2DM) and Parkinson's disease (PD). Efficacy of several antidiabetic drugs has been tested in PD. Exenatide, a synthetic version of exendin-4, an incretin-mimetic drug, is an agonist of glucagon-like peptide 1 receptor (GLP1R) and is approved for the treatment of T2DM. Exenatide can cross the blood-brain barrier and exerts neuroprotective and neurorestorative effects via GLP1R at doses similar to those used in T2DM, resulting in improved motor performance, behaviour, learning and memory in different rodent PD models. Reports in human PD patients have also shown promise. In this work, we carried out substitution at the fourteenth position of exenatide (M14) with basic, acidic and nonpolar residues and investigated their effect on aggregation of recombinant human α-synuclein in vitro and in SH-SY5Y cells. Molecular dynamic (MD) simulation studies showed altered stability of α-synuclein upon substitution at M14 in exenatide. Exenatide had no effect on aggregation of α-synuclein in vitro. The M14K mutant, which stabilized α-synuclein, prolonged lag time and caused significant reduction in aggregation. On the contrary, aggregation of α-synuclein was significantly attenuated in SH-SY5Y cells in the presence of exenatide for all mutants tested, with a concomitant increase in cell survival. Flow cytometric analysis suggested induction of autophagy in the presence of the peptides, explaining the reduction in protein aggregation. Thus, mutants of exenatide could be investigated further as inhibitors of aggregation of α-synuclein.

Mycobacterial infections remain a global public health challenge. Each year, high rates of morbidity and mortality worldwide are a consequence of chronic respiratory infections due to Mycobacteria. According to the World Health Organization (WHO), in 2023, 10.8 million individuals fell ill with Mycobacterium tuberculosis (Mtb), resulting in an estimated 1.25 million deaths. This positions tuberculosis (TB) as the leading cause of death from a single pathogen worldwide after the coronavirus disease (COVID-19) pandemic. On the other hand, the cases of people affected by nontuberculous mycobacteria (NTM) have risen globally, but the precise incidence and prevalence of both pulmonary and extrapulmonary disease remain unknown. In Europe, nontuberculous mycobacterial pulmonary diseases affect between 0.2 and 2.9 per 100 000 individuals, mainly patients with cystic fibrosis (CF) and non-CF bronchiectasis. The diagnosis and treatment of mycobacterial infections are challenging and complex, frequently requiring long-duration treatments with several antibiotics, which in most cases leads to poor patient outcomes. As the role of immune cells has been extensively assessed, in this Review, we summarize the current knowledge about the contribution of epithelial cells in the early steps of Mycobacteria infections. Additionally, we describe how human lung organoid technology provides new tools to better understand host-Mycobacteria interactions in the airways and test new therapeutic targets.

Protein import across the mitochondrial inner membrane typically depends on two protein translocases of the inner membrane (TIM) complexes and the membrane potential. The protozoan parasite Trypanosoma brucei, however, has a single, divergent TIM complex. Unlike other trypanosomal TIM subunits, TbTim20 is not essential for the normal growth of the insect or bloodstream forms of T. brucei, leaving its role uncertain. Specific mutations in the γ-subunit of the F₁F_O-ATPase, such as γL262P, permit bloodstream form trypanosomes to grow without mitochondrial DNA (kinetoplast or kDNA). Here, we show that RNAi-mediated depletion of TbTim20 inhibits growth of this cell line, but only if it lacks the kDNA. Titration of mitochondrial uncouplers and direct membrane potential measurements reveal that TbTim20 becomes more critical as the membrane potential decreases across all tested cell lines. Proteomic analysis of the uninduced and induced γL262P TbTim20-RNAi cell line, which lacks kDNA and exhibits the lowest membrane potential, shows depletion of a subset of imported proteins. This subset includes ATPase subunits, suggesting a mechanism by which TbTim20-silenced cell lines become more sensitive to uncouplers. Thus, we propose that TbTim20 supports the import of a subset of proteins whose import is hypersensitive to a low membrane potential.

Isoprenoid quinones constitute a class of redox lipids that are indispensable for electron transfer in a variety of cellular functions. For instance, plastoquinone, an integral component of plants, algae and Cyanobacteriota, plays a pivotal role in photosynthesis. Isoprenoid quinones are biosynthesised via evolutionary-related pathways, in which some steps are still incompletely characterised. In this study, we confirm the identity of the PlqH enzyme, a flavin-dependent monooxygenase (FMO) conserved in photosynthetic cyanobacteria, which possesses a regioselective hydroxylase activity required for plastoquinone biosynthesis. Phylogenetic analyses demonstrate that cyanobacterial PlqH homologues originated from FMOs involved in bacterial ubiquinone biosynthesis. The synthesis of plastoquinone by Escherichia coli was achieved by expressing two heterologous genes in a genetically engineered strain, which was optimised to produce plastoquinone levels comparable to those of natural ubiquinone. However, plastoquinone was unable to replace ubiquinone in several cellular processes in E. coli, suggesting that fine structural and thermodynamic constraints both play a significant role in the function of quinones.

Acetyl xylan esterases (AcXEs) are crucial for biomass degradation. However, the catalytic mechanism underlying the highly specific activity remains poorly understood, limiting their rational engineering. Here, we characterized the previously undescribed carbohydrate esterase family (CE) 7 acetyl xylan esterase (LaCE7A) from Lactococcus lactis with high specific activity (154 179 U·mg^-1) to unravel the mechanism underlying its efficient catalysis. The monomer structure of LaCE7A presented a typical α/β-hydrolase fold and contained three distinct structure features in CE family 7, exhibiting evolutionary conservation. The large number of hydrophilic residues in the active pocket may increase the affinity of the reaction intermediates to hydrophilic substrates, thus facilitating substrate binding of LaCE7A, which may further contribute to the high specific activity observed. MD simulations indicated that the flexibilities of two regions (residues 139-144 and residues 220-223) increased the volume of the active pocket of the enzyme and were conducive to substrate binding and catalytic reaction. In addition, the unique Ala220 located in the tight turn region of three-helix insertion domain adopted the more favorable trans peptide bond, which played a crucial role in substrate catalysis. Our findings not only identify LaCE7A as a potent biocatalyst but also provide new mechanistic insights into the high activity of CE7 AcXEs, offering a foundation for future enzyme design.

Pyridoxal 5'-phosphate (PLP), the coenzyme form of vitamin B₆, is indispensable for diverse metabolic processes, especially amino acid metabolism. In mammals, PLP is primarily synthesized via a salvage pathway involving pyridoxal kinase (PLK), pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO), and pyridoxal phosphate phosphatase (PLPP). However, recent evidence suggests the presence of additional, yet unidentified, enzymatic contributors to this pathway. Here, we identify aldo-keto reductase family 1 member C (AKR1C) isozymes as previously unrecognized enzymes involved in vitamin B₆ metabolism. We demonstrate that AKR1Cs catalyze two novel reactions: an NADPH-dependent pyridoxal reductase (PLR) activity that converts pyridoxal (PL) to pyridoxine (PN), and an NADP⁺-dependent pyridoxal dehydrogenase (PLD) activity that oxidizes PL to 4-pyridoxolactone (4-PLA). Both reactions occur under physiological conditions and significantly impact intracellular vitamin B₆ vitamer profiles. Moreover, we show that elevated PL levels suppress AKR1C activities toward non-B₆ substrates, indicating reciprocal cross-talk between vitamin B₆ metabolism and other AKR1C-dependent metabolic processes. This study expands the current framework of mammalian vitamin B₆ metabolism, highlighting AKR1Cs as metabolic hubs with broad regulatory implications.