Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein-ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA-NPS), equine (ESA-NPS), and leporine (LSA-NPS) determined to 2.58 Å (C2), 2.42 Å (P61), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA-NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA-NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein.

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Serum albumin, the most abundant transport protein of mammalian blood, interacts with various nonsteroidal anti‐inflammatory drugs (NSAIDs) affecting their disposition, metabolism, and excretion. A big group of chiral NSAIDs transported by albumin, profens, is created by derivatives of 2‐arylpropionic acid. The chiral center in the structures of profens is adjacent to the carboxylate moiety and often determines different pharmacological properties of profen enantiomers. This study describes crystal structures of two albumins, isolated from equine and leporine serum, in complexes with three profens: ibuprofen, ketoprofen, and suprofen. Based on three‐dimensional structures, the stereoselectivity of albumin is discussed and referred to the previously published albumin complexes with drugs. Drug Site 2 (DS2) of albumin, the bulky hydrophobic pocket of subdomain IIIA with a patch of polar residues, preferentially binds (S)‐enantiomers of all investigated profens. Almost identical binding mode of all these drugs clearly indicates the stereoselectivity of DS2 towards (S)‐profens in different albumin species. Also, the affinity studies show that DS2 is the major site that presents high affinity towards investigated drugs. Additionally, crystallographic data reveal the secondary binding sites of ketoprofen in leporine serum albumin and ibuprofen in equine serum albumin, both overlapping with previously identified naproxen binding sites: the cleft formed between subdomains IIIA and IIIB close to the fatty acid binding site 5 and the niche created between subdomains IIA and IIIA, called fatty acid site 6.

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate (Glu)-linked reversible transamination reaction. This process proceeds through a bimolecular ping–pong mechanism and in plants takes place in plastids. It is a part of the phosphorylated pathway of serine biosynthesis, one of three routes recognized in plant organisms that yield serine. In this three-step biotransformation, 3-phosphoglycerate (3-PGA) delivered from plastidial glycolysis and Calvin cycle is oxidized by 3-PGA dehydrogenase. Then, 3-PHP is subjected to transamination with Glu to yield PSer and α-ketoglutarate (AKG). In the last step of the pathway, serine is produced by the action of phosphoserine phosphatase. Here we present the structural characterization of PSAT isoform 1 from Arabidopsis thaliana (AtPSAT1), a dimeric S-shaped protein that truncated of its 71-residue-long chloroplast-targeting signal peptide. Three crystal structures of AtPSAT1 captured at different stages of the reaction: (i) internal aldimine state with PLP covalently bound to the catalytic K265, (ii) holoenzyme in complex with pyridoxamine-5′-phosphate (PMP) after transfer of the amino group from glutamate and (iii) the geminal diamine intermediate state wherein the cofactor is covalently bound to both, K265 and PSer. These snapshots over the course of the reaction present detailed architecture of AtPSAT1 and allow for the comparison of this plant enzyme with other PSATs. Conformational changes of the protein during the catalytic event concern (i) the neighborhood of K265 when the amino group is transferred to the cofactor to form PMP and (ii) movement of the gate-keeping loop (residues 391–401) upon binding of 3-PHP and PSer. The latter conformational change of the loop may likely be one of key elements that regulate catalytic activity of PSATs.

The binding modes to equine serum albumin (ESA) of two nonsteroidal anti-inflammatory drugs (NSAIDs), diclofenac (Dic) and naproxen (Nps), were studied by X-ray crystallography and isothermal titration calorimetry. On the basis of the crystal structure of ESA/Dic determined to a resolution of 1.92 Å and the structure of the previously described ESA/Nps complex (2.42 Å), it was found that both NSAIDs bind within drug site 2 (DS2) of ESA and both occupy secondary binding sites in separate cavities of domain II (Nps) and domain III (Dic). The two structures of the ternary complex ESA/Dic/Nps, obtained by competitive cocrystallization (2.19 Å) and through a displacement experiment (2.35 Å), were determined to investigate possible competition of these widely used pharmaceutical drugs in binding to ESA. In these complexes Nps occupies the DS2 pocket common for both drugs, whereas the other distinct binding sites of Dic and Nps remain unaffected. These results suggest that combined application of both drugs may result in increased concentration of free diclofenac in plasma.

Polyamines are linear polycationic compounds that play a crucial role in the growth and development of higher plants. One triamine (spermidine, SPD) and two tetraamine isomers (spermine, SPM, and thermospermine, TSPM) are obtained by the transfer of the aminopropyl group from decarboxylated -adenosylmethionine to putrescine and SPD. These reactions are catalyzed by the specialized aminopropyltransferases. In that respect, plants are unique eukaryotes that have independently evolved two enzymes, thermospermine synthase (TSPS), encoded by the gene, and spermine synthase, which produce TSPM and SPM, respectively. In this work, we structurally characterize the gene product, TSPS, from the model legume plant (). Six crystal structures of TSPS - one without ligands and five in complexes with either reaction substrate (SPD), reaction product (TSPM), or one of three cofactor analogs (5'-methylthioadenosine,-adenosylthiopropylamine, and adenosine) - give detailed insights into the biosynthesis of TSPM. Combined with small-angle X-ray scattering data, the crystal structures show that TSPS is a symmetric homotetramer with an interdomain eight-stranded β-barrel. Such an assembly and the presence of a hinge-like feature between N-terminal and C-terminal domains give the protein additional flexibility which potentially improves loading substrates and discarding products after the catalytic event. We also discuss the sequence and structural features around the active site of the plant aminopropyltransferases that distinguish them from each other and determine their characteristic substrate discrimination.

The crystal structure of a novel dimeric β-D-galactosidase from Paracoccus sp. 32d (ParβDG) was solved in space group P212121 at a resolution of 2.4 Å by molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

Putrescine, 1,4-diaminobutane, is an intermediate in the biosynthesis of more complexed polyamines, spermidine and spermine. Unlike other eukaryotes, plants have evolved a multistep pathway for putrescine biosynthesis that utilizes arginine. In the final reaction, N-carbamoylputrescine is hydrolyzed to putrescine by N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53). During the hydrolysis, consecutive nucleophilic attacks on the substrate by Cys158 and water lead to formation of putrescine and two by-products, ammonia and carbon dioxide. CPA from the model legume plant, Medicago truncatula (MtCPA), was investigated in this work. Four crystal structures were determined: the wild-type MtCPA in complex with the reaction intermediate, N-(dihydroxymethyl)putrescine as well as with cadaverine, which is a longer analog of putrescine; and also structures of MtCPA-C158S mutant unliganded and with putrescine. MtCPA assembles into octamers, which resemble an incomplete left-handed helical twist. The active site of MtCPA is funnel-like shaped, and its entrance is walled with a contribution of the neighboring protein subunits. Deep inside the catalytic cavity, Glu48, Lys121, and Cys158 form the catalytic triad. In this studies, we have highlighted the key residues, highly conserved among the plant kingdom, responsible for the activity and selectivity of MtCPA toward N-carbamoylputrescine. Moreover, since, according to previous reports, a close MtCPA relative from Arabidopsis thaliana, along with several other nitrilase-like proteins, are subjected to allosteric regulation by substrates, we have used the structural information to indicate a putative secondary binding site. Based on the docking experiment, we postulate that this site is adjacent to the entrance to the catalytic pocket.