Tyrosinase‐related protein 1 (TYRP1) is one of three tyrosinase‐like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase‐like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys‐rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor‐like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism‐related mutations of TYRP1 affect its stability or activity.

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like ( β / α ) ₈ -barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a “U”-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two C—C bonds are broken and concomitantly carbon monoxide is released. The x-ray structures of Aspergillus japonicus 2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-Å resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro ¹⁶⁴ , a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp ³ character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu ⁷³ coordinates the copper through its Oɛ1 atom. The short distance of about 2.55 Å between its Oɛ2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu ⁷³ can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O ₂ binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.

In many Gram-negative pathogens, their virulent behavior is regulated by quorum sensing, in which diffusible signals such as N -acyl homoserine lactones (AHLs) act as chemical messaging compounds. Enzymatic degradation of these diffusible signals by, e.g., lactonases or amidohydrolases abolishes AHL regulated virulence, a process known as quorum quenching. Here we report the first crystal structure of an AHL amidohydrolase, the AHL acylase PvdQ from Pseudomonas aeruginosa . PvdQ has a typical α/β heterodimeric Ntn-hydrolase fold, similar to penicillin G acylase and cephalosporin acylase. However, it has a distinct, unusually large, hydrophobic binding pocket, ideally suited to recognize C12 fatty acid-like chains of AHLs. Binding of a C12 fatty acid or a 3-oxo-C12 fatty acid induces subtle conformational changes to accommodate the aliphatic chain. Furthermore, the structure of a covalent ester intermediate identifies Serβ1 as the nucleophile and Asnβ269 and Valβ70 as the oxyanion hole residues in the AHL degradation process. Our structures show the versatility of the Ntn-hydrolase scaffold and can serve as a structural paradigm for Ntn-hydrolases with similar substrate preference. Finally, the quorum-quenching capabilities of PvdQ may be utilized to suppress the quorum-sensing machinery of pathogens.