Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.     

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.     

A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase

EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70 degrees C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily.The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 A resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical alpha/beta hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors. Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability.     

Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2.

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic mechanism of fungal homoserine transacetylase

Homoserine transacetylase is a required catalyst in the biochemical pathway that metabolizes Asp to Met in fungi. The enzyme from the yeast Schizosaccharomyces pombe activates the hydroxyl group of L-homoserine by acetylation from acetyl coenzyme A. This enzyme is unique to fungi and some bacteria and presents an important new target for drug discovery. Steady-state kinetic parameters provide evidence that this enzyme follows a ping-pong mechanism. Proton inventory was consistent with a single-proton transfer, and pH studies suggested the participation of at least one residue with a pKa value of 6.4-6.6, possibly a His or Asp/Glu in catalysis. Protein sequence alignments indicate that this enzyme belongs to the alpha/beta-hydrolase fold superfamily of enzymes, indicating the involvement of an active-site nucleophile and possibly a canonical catalytic triad. We constructed site-specific mutants and identified Ser163, Asp403, and His432 as the likely active-site residues of a catalytic triad based on steady-state kinetics and genetic complementation of a yeast null mutant. Moreover, unlike the wild-type enzyme, inactive site mutants were not capable of producing an acetyl-enzyme intermediate. Homoserine transacetylase therefore catalyzes the acetylation of L-homoserine via a covalent acyl-enzyme intermediate through an active-site Ser. These results form the basis of future exploitation of this enzyme as an antimicrobial target.     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus

The crystal structure of AFEST, a novel hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus, complexed with a sulphonyl derivative, has been determined and refined to 2.2 A resolution. This enzyme, which has recently been classified as a member of the hormone- sensitive-lipase (H) group of the esterase/lipase superfamily, presents a canonical alpha/beta hydrolase core, shielded on the C-terminal side by a cap region composed of five alpha-helices. It contains the catalytic triad Ser160, His285 and Asp255, whereby the nucleophile is covalently modified and the oxyanion hole formed by Gly88, Gly89 and Ala161. A structural comparison of AFEST with its mesophilic and thermophilic homologues, Brefeldin A esterase from Bacillus subtilis (BFAE) and EST2 from Alicyclobacillus acidocaldarius, reveals an increase in the number of intramolecular ion pairs and secondary structure content, as well as a significant reduction in loop extensions and ratio of hydrophobic to charged surface area. The variety of structural differences suggests possible strategies for thermostabilization of lipases and esterases with potential industrial applications.     

Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.     

Identification and analysis of a new family of bacterial serine proteinases

A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration

Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.     

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.     

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.     

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.     

Completion of the canonical pathway for assembly of anticancer drugs vincristine/vinblastine in Catharanthus roseus

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP‐β‐l ‐rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP‐β‐l ‐rhamnose. The mutant H357Q exhibited activity with both UDP‐β‐l ‐rhamnose and UDP‐glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor‐His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.