Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of d-mannose and d-glucose, has recently been characterized to have potential for d-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME–d-glucitol and RsME(D254A)–d-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7–α8). The RsME–d-glucitol structure showed that loopα7–α8 moves towards d-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7–α8 are only conserved in MEs and interact with d-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)–d-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7–α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a Kd of 0.1 ± 0.01 µM. In contrast, a Kd of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox–DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

A figure in the article by Barbarin-Bocahu & Graille [(2022), Acta Cryst. D78, 517–531] is corrected.

l-Proline hydroxylase is a member of the non-heme Fe2+/α-ketoglutarate (AKG)-dependent hydroxylase family that catalyzes the reaction from l-proline to hydroxy-l-proline, which is widely used in drug synthesis, biochemistry, food supplementation and cosmetic industries. Here, the first crystal structure of l-proline trans-hydroxylase and its complexes with substrate and product are reported, which reveal the structural basis of trans–cis proline hydroxylation selectivity. Structure comparison with other AKG-dependent hydroxylases identifies conserved amino acid residues, which may serve as signatures of in-line or off-line AKG binding modes in the AKG-dependent enzyme family.

Celebrating 30 years of Acta D and looking forward to the future of structural biology.

Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 acyl bond of lecithin in a Ca2+-dependent manner, is an important enzyme in the oil and oleochemical industries. However, most sPLA2s are not stable under process conditions. Therefore, a thermostable sPLA2 was investigated in this study. A marine bacterial sPLA2 isolated from Sciscionella marina (Sm-sPLA2) was catalytically active even after 5 h of incubation at high temperatures of up to 50°C, which is outstanding compared with a representative bacterial sPLA2 (i.e. sPLA2 from Streptomyces violaceoruber; Sv-sPLA2). Consistent with this, the melting temperature of Sm-sPLA2 was measured to be 7.7°C higher than that of Sv-sPLA2. Furthermore, Sm-sPLA2 exhibited an improved biotransformation performance compared with Sv-sPLA2 in the hydrolysis of soy lecithin to lysolecithin and free fatty acids at 50°C. Structural and mutagenesis studies revealed that the Trp41-mediated anchoring of a Ca2+-binding loop into the rest of the protein body is directly linked to the thermal stability of Sm-sPLA2. This finding provides a novel structural insight into the thermostability of sPLA2 and could be applied to create mutant proteins with enhanced industrial potential.

Aggregates of the antioxidant superoxide dismutase 1 (SOD1) are one of the major contributors to the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in SOD1 lead to an unstable structure and aggregation that perturbs the balance of reactive oxygen species in cells. Oxidation damage to the solvent-exposed Trp32 also causes aggregation of SOD1. Here, the FDA-approved antipsychotic drug paliperidone is identified to interact with Trp32 of SOD1 by structure-based pharmacophore mapping and crystallographic studies. Paliperidone is used for the treatment of schizophrenia. The crystal structure of the complex with SOD1, refined to 2.1 Å resolution, revealed that the ligand binds to the SOD1 β-barrel in the β-strand 2 and 3 regions, which are known to scaffold SOD1 fibrillation. The drug also makes substantial π–π interaction with Trp32. Microscale thermophoresis studies confirm significant binding affinity of the compound, suggesting that the ligand can inhibit or prevent tryptophan oxidation. Thus, the antipsychotic drug paliperidone or a derivative may avert SOD1 aggregation and can be used as a lead for ALS drug development.

The bromodomain and extra-terminal (BET) family proteins, which are involved in chromatin function, have been shown to be promising drug targets in several pathological conditions, including cancer and inflammation. There is considerable interest in the development of BET inhibitors with novel scaffolds to modulate the epigenesis of such diseases. Here, high-resolution crystal structures of the purine class of FDA-approved drugs (theophylline, doxophylline and acyclovir) and non-FDA-approved compounds (3-methyl-7-propylxanthine and theobromine) complexed with hBRD2 bromodomains BD1 and BD2 are reported. Remarkably, a new binding site is exhibited by stacking the compounds against the WPF shelf of BD1 and BD2. This serendipitous binding, in addition to the known acetyl-lysine binding site, sufficiently anchors the ligands in the solvent-exposed region. In addition, slight variations in the lipophilicity of these molecules significantly affected the in vitro binding affinity and selectivity towards BD1 compared with BD2. This idiosyncratic binding provides a new structural framework to link these sites for the development of next-generation inhibitors of the BET family.