John F. Cutfield

Identification of Glu-330 as the Catalytic Nucleophile of Candida albicans Exo-β-(1,3)-glucanase

The exo-β-(1,3)-glucanase from Candida albicans hydrolyzes cell wall β-glucans via a double-displacement mechanism involving a glycosyl enzyme intermediate. Reaction of the enzyme with 2′,4′-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside resulted in the time-dependent inactivation of this enzyme via the accumulation of a 2-deoxy-2-fluoro-glycosyl-enzyme intermediate as monitored also by electrospray mass spectrometry. The catalytic competence of this intermediate is demonstrated by its reactivation through hydrolysis (kreact = 0.0019 min−1) and by transglycosylation to benzyl thio-β-D-glucopyranoside (kreact = 0.024 min−1; Kreact = 56 mM). Peptic digestion of the labeled enzyme followed by tandem mass spectrometric analysis in the neutral loss mode allowed detection of two glycosylated active site peptides, the sequences of which were identified as NVAGEW and NVAGEWSAA. A crucial role for Glu-330 is confirmed by site-directed mutagenesis at this site and kinetic analysis of the resultant mutant. The activity of the Glu-330 → Gln mutant is reduced over 50,000-fold compared to the wild type enzyme. The glutamic acid, identified in the exoglucanase as Glu-330, is completely conserved in this family of enzymes and is hereby identified as the catalytic nucleophile.

The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY

Two pancreatic peptides, somatostatin‐28 and peptide YY, have been isolated from the Brockmann bodies of the teleost fish Cottus scorpius (daddy sculpin). Following purification by reverse‐phase HPLC, each peptide was sequenced completely through to the carboxyl‐terminus by gas‐phase Edman degradation. Somatostatin‐28 was the major form of somatostatin detected and is similar to the gene II product from angler‐fish. Peptide YY (36 amino acids) more closely resembles porcine neuropeptide YY and intestinal peptide YY than it does the pancreatic polypeptides.

Low resolution crystal structure of hagfish insulin

Abstract Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P41212 with a monomer in the asymmetric unit. The application of the Rossmann & Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 A resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.

Identification of a putative active site residue in the exo‐β‐(1,3)‐glucanase of Candida albicans

Recombinant exo‐β‐(1‐3)‐glucanase from Candida albicans was expressed in Saccharomyces cerevisiae and purified. The enzyme contains a number of short blocks of sequence homology with several genes for cellulases of the family A glycanases including the conserved sequence motif NEP which has previously been shown to be important in the catalytic function of several cellulases. Site directed mutagenesis of this glutamic acid residue in the 1,3 glucanase (E230D, E230Q) decreased the enzymatic activity 15,000‐ and 400‐fold, respectively. This suggests that the E of the NEP participates in catalysis of the exoglucanase and other related glycanases.

Biophysical Characterization of ERp29 EVIDENCE FOR A KEY STRUCTURAL ROLE OF CYSTEINE 125

ERp29 is a major resident of the endoplasmic reticulum (ER) that seemingly plays an important role in most animal cells. Although a protein-folding association is widely supported, ERp29s specific molecular function remains unknown. A chaperone activity was postulated from evidence that ERp29 forms multimers like the classical ER chaperones, but conflicting results have emerged from our recent studies. Here a biophysical approach was used to clarify this issue and also reveal a key structural role for ERp29s characteristic cysteine, Cys-125. Applying hydrodynamic parameters derived from sedimentation and dynamic light-scattering analyses, a model of ERp29s quaternary structure was assembled from existing tertiary substructures. Comparison with Windbeutel, an ERp29-like protein from fruit fly with specialized chaperone activity, revealed similar tri-lobar gross structures but some finer differences consistent with functional divergence. Solubility and hydrophobic probe assays revealed moderate surface hydrophobicity, which was reduced in mutant ERp29 in which serine replaced Cys-125. This mutant was also relatively labile to proteolytic degradation, providing two reasons for the strict conservation of Cys-125. No multimerization was observed with untagged ERp29, which existed as tight homodimers (Kd < 50 nm), whereas His-tagged ERp29 artifactually formed 670-kDa oligomers. These findings distinguish ERp29 biophysically from its peers in the ER including Windbeutel, endorsing our postulate that ERp29 adds a distinct type of folding activity to the ER machinery. By invoking novel functional associations for Cys-125 and the adjoining linker, new clues about how ERp29 might work have also arisen.

Structure and activity of exo-1,3/1,4-β-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain

Following the discovery of an exo‐1,3/1,4‐β‐glucanase (glycoside hydrolase family 3) from a seaweed‐associated bacterium Pseudoalteromonas sp. BB1, the recombinant three‐domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two‐domain barley homologue β‐d‐glucan exohydrolase (ExoI) with a distinctive Trp–Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β‐glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β‐sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A–ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small‐angle X‐ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate‐binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate.

Discovery and Characterization of a Distinctive Exo-1,3/1,4-β-Glucanase from the Marine Bacterium Pseudoalteromonas sp. Strain BB1

ABSTRACT Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-β-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-β-d-glucan with some 1,6-β branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-β-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-β-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-β linkages and 1,3-β linkages, and so ExoP is designated an exo-1,3/1,4-β-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.

Structure of Secreted Aspartic Proteinases from Candida

Pathogens of the genus Candida can cause life threatening infections in immunocompromised patients. The three–dimensional structures of two closely related secreted aspartic proteinases from C. albicans complexed with a potent (Ki=0.17 nM) inhibitor, and an analogous enzyme from C. tropicalis reveal variations on the classical aspartic proteinase theme that dramatically alter the specificity of this class of enzymes. The novel fungal proteases present: i) an 8 residue insertion near the first disulfide (Cys45–Cys50, pepsin numbering) that results in a broad flap extending towards the active site; ii) a seven residue deletion replacing helix hN2 (Serll0–Tyrll4), which enlarges the S3 pocket; iii) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and iv) an ordered 12 residue addition at the car–boxy terminus. The same inhibitor (A–70450) binds in an extended conformation in the two variants of C albicans protease, and presents a branched structure at the P3 position. However, the conformation of the terminal methylpiperazine ring is different in the two crystals structures. The implications of these findings for the design of potent antifungal agents are discussed.

Carbohydrate binding sites in Candida albicans exo-β-1,3-glucanase and the role of the Phe-Phe 'clamp' at the active site entrance.

Candida albicans exo‐β‐1,3‐glucanase (Exg; EC 3.2.1.58) is implicated in cell wall β‐d‐glucan remodelling through its glucosyl hydrolase and/or transglucosylase activities. A pair of antiparallel phenylalanyl residues (F144 and F258) flank the entrance to the active site pocket. Various Exg mutants were studied using steady‐state kinetics and crystallography aiming to understand the roles played by these residues in positioning the β‐1,3‐d‐glucan substrate. Mutations at the Phe‐Phe entranceway demonstrated the requirement for double‐sided CH/π interactions at the +1 subsite, and the necessity for phenylalanine rather than tyrosine or tryptophan. The Tyr‐Tyr double mutations introduced ordered water molecules into the entranceway. A third Phe residue (F229) nearby was evaluated as a possible +2 subsite. The inactive double mutant E292S/F229A complexed with laminaritriose has provided the first picture of substrate binding to Exg and demonstrated how the Phe‐Phe arrangement acts as a clamp at the +1 subsite. The terminal sugar at the −1 site showed displacement from the position of a monosaccharide analogue with interchange of water molecules and sugar hydroxyls. An unexpected additional glucose binding site, well removed from the active site, was revealed. This site may enable Exg to associate with the branched glucan structure of the C. albicans cell wall.

Insulin-like meterial from the digestive tract of the tunicate Pyura pachydermatina (sea tulip)

Gut tissue from the tunicate Pyura pachydermatina (sea tulip) was found to contain a compound or compounds which react with anti-porcine insulin antibodies, but not anti-hag-fish insulin antibodies, and which also stimulate lipogenesis in isolated rat fat cells. The insulin-like material is present in two immunologically active forms, a species of Mr 6000 apparently similar to mammalian insulin, and a high Mr form which expresses biological activity only after further purification by reverse-phase HPLC. The bioactivity of both species is suppressed in the presence of anti-porcine insulin antibodies.