The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment. While the structures of other fungal and yeast glucoamylase catalytic and starch binding domains have been determined separately, this is the first intact structure that allows visualization of the juxtaposition of the starch binding domain relative to the catalytic domain. The detailed interactions we see between the catalytic and starch binding domains are confirmed in a second independent structure determination of the enzyme in a second crystal form. This second structure model exhibits an identical conformation compared to the first structure model, which suggests that the H. jecorina glucoamylase structure we report is independent of crystal lattice contact restraints and represents the three-dimensional structure found in solution. The proposed starch binding regions for the starch binding domain are aligned with the catalytic domain in the three-dimensional structure in a manner that supports the hypothesis that the starch binding domain serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T m ) of 68.7°C, 14.3°C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T m 9.1°C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T m 3.9°C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.Keywords: Thermal stability; cellulase; cellulose; endoglucanase; homolog; protein crystal structure Bacterial and fungal cellulases are widely used in the detergent, textile, and food industries, and there is continuing interest in their potential use in the conversion of cellulosic biomass to fermentable sugars. Cellulases are glycoside hydrolases found in at least 12 families of this very large group of enzymes (Henrissat and Davies 2000), and consist of cellobiohydrolases (or exoglucanases, EC 3.2.1.91) and endoglucanases (EC 3.2.1.4). Glycoside hydrolase family 12 (GH 12) members hydrolyse the ␤-1,4-glycosidic bond in cellulose via a double displacement reaction and a glycosylenzyme intermediate that results in retention of the anomeric configuration in the product (Schulein 1997;Birsan et al. 1998). Structures of GH 12 endoglucanases from both bacterial (Sulzenbacher et al. 1997;Crennell et al. 2002) and fungal (Sandgren et al. 2001(Sandgren et al. , 2003Khademi et al. 2002) sources have now been determined, and these provide the structural framework for the whole family. As part of a program to discover cellulases with improved properties, many novel GH 12 endoglucanases have been cloned and Abbreviations: CD, circular dichroism; DTNB, 5,5Ј-dithiobis-2-nitrobenzoic acid; GH, glycoside hydrolase; HPLC, high-pressure liquid chromatography; mme, mono-methyl-ether; NCS, noncrystallographic symmetry; PEG, polyethylene glycol; RMSD, root-mean-square deviation; TFA...

PDB Reference: chymotrypsin, 2ea3, r2ea3sf.The crystal structure of a secreted chymotrypsin from the alkaliphile Cellulomonas bogoriensis has been determined using data to 1.78 Å resolution and refined to a crystallographic R factor of 0.167. The crystal structure reveals a large P1 substrate-specificity pocket, as expected for chymotrypsins. The structure is compared with close structural homologues. This comparison does not reveal clear reasons for the alkali tolerance of the enzyme, but the greater compactness of the structure and lowered hydrogen bonding may play a role.

A new device for rapid enzymatic debridement of cutaneous wounds has been developed using a controlled-release, silicone-based, dried emulsion. A dehydrated serine protease of the subtilisin family, previously untested for wound debridement, was incorporated into the emulsion. This device exhibited excellent storage stability. Moisture from the wound triggered an even, reproducible, and complete release of the enzyme within the first 8 hours. The device maintains a moist wound environment that allows the enzyme to achieve nearly complete digestion of the hardened eschar of full-thickness burns in a porcine model after an exposure period of 24 hours. Debridement was faster than in untreated wounds or wounds treated with a currently available enzyme ointment. Following rapid enzymatic debridement, healing appeared to progress normally, with no histological evidence of damage to adjacent healthy tissue.