Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the βκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the βκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing βκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Sulfated fucans attract increasing research interests in recent decades for their various physiological activities. Fucanases are indispensable tools for the investigation of sulfated fucans. Herein, a novel GH168 family endo-1,3-fucanase was cloned from the genome of marine bacterium Wenyingzhuangia fucanilytica. The expressed protein Fun168D was a processive endo-acting enzyme. Ultra performance liquid chromatography-high resolution mass spectrum and NMR analyses revealed that the enzyme cleaved the α-1 → 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2OSO3-) in sulfated fucan from Isostichopus badionotus, and α-1 → 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2,4OSO3-) in sulfated fucan from Holothuria tubulosa. Fun168D would prefer to accept α-l-Fucp(2,4OSO3-) than α-l-Fucp(2OSO3-) at subsite +1, and could tolerate the absence of fucose residue at subsite +2. The novel cleavage specificity and hydrolysis pattern revealed the presence of diversity within the GH168 family, which would facilitate the development of diverse biotechnological tools for the molecule tailoring of sulfated fucan.

OBJECTIVE: This study presents a novel classification of the anatomical subsites of the tonsillar fossa and discusses their associations with post-tonsillectomy hemorrhage (PTH) after extracapsular tonsillectomy.
METHODS: Coblation tonsillectomy was performed on three adult cadavers and the anatomical subsites of the tonsillar fossa based on the distribution of the tonsillar feeding artery: the upper pole (subsite A), most of the tonsil body (subsite B), the inferior tonsil body (subsite C), and components of the lower pole (subsites D and E). Extracapsular tonsillectomy was prospectively performed using various surgical techniques and PTH was evaluated.
RESULTS: A cadaveric study revealed that the intra- and extra-capsular vessel topographies were essentially identical. Although the demarcation lines varied either up or down by a few millimeters, the arterial vascular network was particularly dense at subsites D and E, and the vessel diameter at these subsites was significantly greater than at subsite C and also (especially) at subsite E. Of 680 patients who underwent tonsillectomy, PTH developed early in 13 (31.7%) and late in 28 (68.3%). Surgical interventions were required by 29/41 patients (70.7%). Subsites D and E were the most common subsites of late PTH and PTH that required intervention. Such intervention was rarely necessary when PTH developed at subsite A or B.
CONCLUSIONS: The new classification of the anatomical subsites of the tonsillar fossa aids inexperienced surgeons and provides an anatomical rationale for variation in surgical technique that minimizes vascular injury, thus improving safety.

Agarans are sulfated galactans extracted from red algae with high structural complexity, of which natural methylation often occurs on the O-6 position of its β-d-galactopyranose units. Although many agaran degrading enzymes, including agarases and porphyranases, have been characterized, little attention has been paid to the tolerance of methyl groups at cleavage subsites. In this study, the structure of GH86 β-agarase Aga86A_Wa from Wenyingzhuangia aestuarii was determined by X-ray crystallography and investigated from a structural biology perspective. The structure indicated that an accommodation pocket formed by F367, Y280, and Q326 at subsite -1 contributes to the methyl-galactose tolerance of Aga86A_Wa. Furthermore, we found that similar accommodation pockets were present in the structures of two other GH86 enzymes BuGH86 from Bacteroides uniformis and BpGH86A from Phocaeicola plebeius, and their previously undisclosed methyl-galactose tolerance was verified, validating the function of the pockets. Phylogenetic analysis, structural modeling, and hydrolysis product characterization suggested that the methyl-galactose accommodation capacity at subsite -1 was prevalent in GH86 members. These findings achieve a better understanding of the function and mechanism of GH86 agaran degrading enzymes, and will facilitate the precise preparation of agaran oligosaccharides by employing defined tools.

BACE1 is a key aspartic protease that cleaves the amyloid precursor protein to generate of the amyloid peptide that is believed to be responsible for the Alzheimer\'s disease amyloid cascade. It is thus recognized as a promising therapeutic target for Alzheimer\'s disease treatment, and large efforts have been made in the discovery of novel BACE1 inhibitors. This Review presents a systematic mining of BACE1 inhibitors based on 354 crystal structures of the BACE1 catalytic domain in complex with ligands in the Protein Data Bank. A thorough exploration on the frequency as well as the patterns of residue-ligand interactions enables us to subdivide the ligand binding pocket into 10 subsites and then identify favorable substructures of ligands for each subsite. In addition, it is found that the assembly of subsites with an 8-like shape is responsible to bind all inhibitors and four major ligand binding modes are revealed. Thus, such a systematic survey deepens our understanding of the structural requirements for establishment of BACE1-ligand interactions that determine the affinity of a ligand to BACE1, which is pivotal for structure-based lead optimization and design of novel inhibitors.

Here we report the first crystal structure of a secretory α-glucoside hydrolase isolated from Pseudoalteromonas sp. K8, PspAG97A, which belongs to glycoside hydrolase family 97 and exhibits halophilic property. PspAG97A lacks an acidic surface, that is considered essential for protein stability at high salinity. Interestingly, PspAG97A unusually contains a chloride ion coordinated by the guanidinium group of Arg171 and the main chain amide groups of Tyr172 and Glu173 at the active site. The structures of PspAG97A complexed with acarbose and panose demonstrate that residues Glu173, Arg171 and Asn170 for subsite +1 decide the substrate specificity of the enzyme for the α-1,6-glucosidic linkage. Structural alterations observed in the R171K variant and enzyme kinetic experiments focusing on chloride assisted activation suggest that the active site chloride serves to properly orient Glu173, Arg171 and Asn170 to facilitate substrate recognition. Furthermore, the chloride assists the binding of Glu173 to the conserved calcium ion and plays an essential role in properly positioning the base catalyst Glu456. In sum, our results provide valuable insight into the structural basis of protein halophilicity.