Protein structure and structure/function relationships

J. Le Nours; C. Ryttersgaard; L.L. Leggio; P.R. Østergaard; T.V. Borchert; L.L.H. Christensen; S. Larsen.

"Structure of two fungal beta-1,4-galactanases: Searching for the basis for temperature and pH optimum."

Protein Science, 12, 1195-1204 (2003)

Abstract
beta-1, 4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1, 4- galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the Gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing similar to56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 Angstrom and 2.14 Angstrom resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 Angstrom resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the a-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/H91D mutant of MTGAL showed that th e difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

W. Receveur; M. Czjzek; M. Schulein; P. Panine; B. Henrissat.

"Dimension, shape, and conformational flexibility of a two domain fungal cellulase in solution probed by small angle X- ray scattering."

J. Biol. Chem., 277(43), 40887-40892 (2002)

Abstract
Cellulase Ce145 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Ce145 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.

J. E. Nielsen; T. V. Borchert; G. Vriend.

"The determinants of alpha-amylase pH activity profiles."

Protein engineering, 14, 505-512 (2001)

Abstract
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha -amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 alpha -amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the pK(a) values of the active site residues. More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha -amylases.

"Oligosaccharide binding to family 11 xylanases: both covalent intermediate and mutant product complexes display B-2,B-5 conformations at the active centre."

Acta Crystallographica Section D-Biological Crystallography, 57, 1344-1347 (2001)

Abstract
The glycoside hydrolase sequence-based classification reveals two families of enzymes which hydrolyse the beta -1, 4-linked backbone of xylan, xylanases, termed families GH-10 and GH- 11. Family GH-11 xylanases are intriguing in that catalysis is performed via a covalent intermediate adopting an unusual B-2, B-5 (boat) conformation, a conformation which also fulfils the stereochemical constraints of the oxocarbenium ion-like transition state. Here, the 1.9 Angstrom structure of a nucleophile, E94A, mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose is presented. Intriguingly, this complex also adopts the B-2, B-5 conformation in the -1 subsite, with the vacant space provided by the Glu --> Ala mutation allowing the sugar to adopt the alpha -configuration at C1. The structure of the covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate has been extended to atomic (1.1 Angstrom) resolution.

K. Zhu; A. Jutila; E.K.J. Tuominen; S.A. Patkar; A. Svendsen; P.K.J. Kinnunen.

"Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability."

Biochim. Biophys. Acta - Protein Structure and Molecular Enzymology, 1547, 329-338 (2001)

Abstract
Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T-d = 74.4 degreesC whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degreesC, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T-m) at approx. 70.5, 61.0, and 65.5 degreesC for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However. faster mobilities became evident when the unfolding temperatures (T-m) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degreesC) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL. with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations. especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

L. Beier; A. Svendsen; C. Andersen; T. P. Frandsen; T. V. Borchert; J.R. Cherry.

"Conversion of the maltogenic alpha-amylase into a CGT'ase."

Protein Engineering ,13,  509-513 (2000)

Abstract
Novamyl is a thermostable five-domain maltogenic alpha- amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases), Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce beta- CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five- residue loop deleted was not able to form beta-CD.

A.M. Brzozowski; H. Savage; C.S. Verma; J.P.D.M. Lawson; A. Svendsen; S.A. Patkar.

"Structural origins of the interfacial activation on Thermomyces (Humicola) lanuginosa lipase."

Biochemistry, 39,15071-15082 (2000 )

Abstract
The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TIL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha (0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TIL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.