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New amylases through gene manipulation designed by crystallography, structure comparison and affinity labeling

Objective

to design and produce enzyme variants that degrade starch and related saccharides with modified specificity. The goal is achieved through protein engineering and crystallography of barley alpha-amylase (AMY1 and 2), cyclodextrinase, isoamylase, glucoamylase and its starch binding domain (SBD).
AMY1 action pattern and specificity were engineered by random mutagenesis of RGY185 and FVD287 at the beta->alpha 4 and 7 substrate binding loops of the (beta/alpha)8-fold. Three and two-fold activity increase was obtained on either oligosaccharides or amylose, as seen for KGY185 and SGM185, respectively. Loss of a H-bond to substrate at subsite +2 shifted the product profile. Loop 7 mutants show increased relative specificity for short over long substrates and altered product profile. Based on subsite +4 proposed to be at beta->alpha 6 by photoaffinity labelling, an AMY2->AMY1 tetrapeptide replacement was made. This variant has action pattern intermediary to the parents. AMY1-AMY2 hybrids, generated by homeologous recombination in yeast, have improved enzymatic properties. Moreover, the origin of domain B controls isozyme specific properties, incl. inhibition of AMY2 by barley a-amylase/subtilisin inhibitor, BASI. Domain B mutants probed binding contributions by AMY2 side-chains and acquired BASI inhibition of AMY1. The crystal structure of BASI/AMY2 is solved and the BASI/AMY2/subtilisin complex was generated by modeling.Wt, Delta405-414 (matching the AMY2 length), and SGM185-AMY1, produced in Pichia pastoris, gave diffracting flat, bad flat, and yet no crystals. The former diffract to 3 Å resolution. One dimension must be enlarged. Promising small crystals made in gels will be examined at the synchrotron. The 80% sequence identity allowed modeling of AMY1 guided by the 3D of AMY2. Notably differences occur in domain B, AMY1 having Cys95 (Thr95 in AMY2) in an S-S bond to Cys106 and only 2 Pro, compared to 5 in AMY2.
A fusion gene encoding AMY1-SBD was constructed by overlap extension PCR, incl. the full length linker from A. niger glucoamylase. The fusion and the AMY1 cDNA were inserted in A. niger pAN52 between the GPD promoter and trpC terminator for transformation of A. niger AB4-1. AMY1-SBD and AMY1 were effectively secreted when using the AMY1 native signal yielding up to 100 mg/L (AMY1). SDS-PAGE, Western blot, N-terminal sequence, and activity analysis confirmed that correctly processed, functional proteins were produced. Interactions of the AMY1-SBD with insoluble starches and heterbifunctional inhibitors are currently examined.
An intramolecularly fluorescence-quenched amylase substrate was synthesized using Taka-amylase for transglycosylation. Thiooligosaccharides substrate analogues are synthesized by a telomerization procedure. The synthesis failed of those with exclusively thioglycosidic linkages. A disaccharide synthon was prepared. The synthesis of 4{w}-S-alpha-D-glucopyranosyl-4{w}-thiomaltooligosaccharides was achieved of DP 4 and 5 using alpha-amylase. These analogues are competitive inhibitors of glucoamylase. The synthesis of cyclothiomaltin and tri-deoxy-alpha-cyclodextrin was successful through the effective conversion catalyzed by CGTase from B. circulans of 4-thio and 6'-iodo-alpha-maltosyl fluorides. Both sets of compounds are potential inhibitors for alpha-amylase and CGTase. Crystal structures of CGTase in complex with cyclothiomaltins or a branched 6-S-Glc-beta-CD was determined. In the latter case all b-CD binding subsites were defined in the structure. Bifunctional probes targeted to the active site and the starch binding domain (SBD) of glucoamylase G1 were synthesized by coupling acarbose, a potent inhibitor of G1, to a monomodified cyclodextrin through oligoethylene-glycol spacers. These compounds trap glucoamylase G1 in a closed conformation as determined by dynamic light scattering experiments and titration calorimetry. Homologue replacement of glucoamylase binding loops shifted the bond-type relative specificity.
Crystals are obtained of cyclodextrinase from Flavobacterium sp. no. 92 which was cloned and sequenced and expressed in E. coli (50 mg/L). Of a dozen different forms, three were characterized in detail by X-ray diffraction. One grew to suitable sizes and diffracted beyond 3 Å resolution after shock freezing to 100 oK. There are 8 molecules in the asymmetric unit in the space group C2. This packing constitutes a tremendous X-ray analytical problem. Various attempted heavy atom derivatizations were unsuccessful. Because the enzyme in the future will be engineered, surface segments were tentatively identified and subjected to mutation to obtain a new crystal packing. Finally, a data base of pyranose saccharide binding sites has been established.

MAJOR SCIENTIFIC BREAKTHROUGHS:
protein engineered barley alpha-amylase (beta/alpha)8-barrel beta->alpha mutants were obtained by random mutagenesis and screening with superior activity and altered action pattern on short maltooligodextrins. AMY1-AMY2 hybrids had improved properties relative to both parent isozymes. AMY1 wt and variants, obtained at 50 mg/L in P. pastoris, crystallize. A functional fusion AMY1-SBD is produced (50 mg/L) in A. niger. Modulation is obtained by homologue loop replacement of the glucoamylase bond-type specificity. Suitable crystals are obtained of cyclodextrinase from Flavobacterium sp.. The linker-connected catalytic and starch binding domains of glucoamylase G1 are close in solution as probed by novel spacer-linked synthetic heterobifunctional inhibitors. For the first time all glucosyl residues are defined in the 3D structure of CGTase by using a synthetic beta-CD derivative.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

CARLSBERG LABORATORY
Address
10,Gamle Carlsberg Vej 10
2500 Valby
Denmark

Participants (4)

Albert-Ludwigs-Universität Freiburg
Germany
Address
Albertstraße 21
79104 Freiburg
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
France
Address
601,Rue De La Chimie 601, Domaine Universitaire
38041 Grenoble
Centre National de la Recherche Scientifique (CNRS)
France
Address
Boulevard Pierre Dramard
13326 Marseille
Institute of Food Research
United Kingdom
Address

NR4 7UA Norwich