Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley β-amylase (original) (raw)
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1998
a-Amylases are widely occurring, multidomain proteins with a catalytic (b/a) 8 -barrel. In barley a-amylase, insight into the catalytic mechanism is gained from the X-ray crystal structure of its molecular complex with acarbose, a pseudotetrasaccharide that acts like a transition-state analogue and which is shown to bind at two speci®c regions of the enzyme. The structure of the complex has been re®ned to an R-factor of 15.1% for all observations with F o > s(F o ) between 10 and 2.8 A Ê resolution. A difference Fourier map produced after re®nement of the native structure against the data of the acarbose complex clearly revealed density corresponding to two oligosaccharide-binding sites. One of these is de®ned as the surface-located starch granule-binding site characteristic of cereal a-amylases. It involves stacking of two acarbose rings on Trp276 and Trp277. The other binding region is the active site covering subsites À1, 1 and 2. Here, Glu204 is positioned to act in general acid/base catalysis protonating the glucosidic oxygen atom assisted by Asp289. A water molecule that bridges Glu204 and Asp289 is found at the entrance cavity containing a total of ®ve water molecules. This water molecule is proposed to reprotonate Glu204 and supply the hydroxyl ion for nucleophilic attack on the glucosyl C1 atom. Asp 179 acts as the nucleophile that can bind covalently to the substrate intermediate after bond cleavage. The present complex structure together with the conservation of active-site residues among a-amylases and related enzymes, are consistent with a common catalytic mechanism for this class of retaining carbohydrases. Abbreviations used: AMY1, barley low-pI a-amylase (isozyme 1); AMY2, barley high-pI a-amylase (isozyme 2); TAA, Taka-amylase A (Aspergillus oryzae); AAA, a-amylase from Aspergillus niger; PPA, pig pancreatic a-amylase; BLA, Bacillus licheniformis a-amylase; CGT, cyclodextrin glucosyltransferase from Bacillus circulans; BASI, barley a-amylase/subtilisin inhibitor; r.m.s., rootmean-square; F o , F c , observed and calculated structure factor amplitudes, respectively; R sym AE|I À hIi|/AEI, where I is the observed intensity and hIi is the average intensity obtained from multiple observations of symmetry-related re¯ections R-factor AE|F o À F c |AEF o .
Interactions between barley. ALPHA.-amylases, substrates, inhibitors and regulatory proteins
J Appl …, 2005
Amylases belong to the glycoside hydrolase family 13 (GH13) that together with glycoside hydrolase families 70 and 77 constitute clan H of glycoside hydrolases (GH H). 1) GH H contains approximately 30 different enzyme specificities acting in hydrolase and transglucosidase reactions on α 1,4 and or α 1,6 glucan and α glucoside substrates. 2) The three dimensional structures of α amylases mostly contain three domains; domain A, a (β α)8 barrel; domain B a small protruding loop situated between β strand and α helix 3 in the (β α)8 barrel; and domain C, a C terminal anti parallel β sheet composed of 5 10 strands. The substrate binding site is made from residues of domains A and B and the three acid residues involved in catalysis are situated at the C terminal ends of β strands 4, 5 and 7. 2) These three catalytic residues are the only invariant residues in GH H. 3) A few new complexes between amylolytic enzymes and substrates or substrate analogues have been recently published. 4,5) These structures provide novel insight in particular with respect to
2002
ROBERT ,X., H ASER ,R., S VENSSON ,B. &A GHAJARI, N., Comparison of crystal structures of barley -amylase 1 and 2: implications for isozyme dif- ferences in stability and activity. Biologia, Bratislava, 57/Suppl. 11: 59|70, 2002; ISSN 0006-3088. The germinating barley seed contains two major -amylase isozyme families AMY1 and AMY2 involved in starch degradation to provide energy used by
European journal of biochemistry, 2001
Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites 25/26 (Cys95 ! Ala/Thr) and þ1/þ 2 (Met298 ! Ala/ Asn/Ser) as well as in the double mutants, Cys95 ! Ala/ Met298 ! Ala/Asn/Ser. Cys95 ! Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl b-D-maltoheptaoside (Cl-PNPG 7), respectively, but fivefold to 20-fold higher K m. The Cys95 ! Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 ! Ala and wild-type. Met298 ! Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k cat and k cat /K m for Cl-PNPG 7 are # 30% and # 10% of wild-type, respectively. The activity of Cys95 ! Ala/Met298 ! Ala/ Asn/Ser is 100-180% towards starch, and the k cat /K m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG 7 , respectively, emphasizing the strong impact of the Cys95 ! Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG 7 and amylose/Cl-PNPG 7 are 2.8-to 270-fold and 1.2-to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites 25 and þ2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites þ1 and þ2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6 000-maltotriosyl-maltohexaose thus covering subsites 2 1 to þ5, while productive binding of unbranched substrate involves subsites 2 3 to þ3.
Multi-site substrate binding and interplay in barley α-amylase 1
FEBS Letters, 2008
Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi-site substrate interactions are functionally significant. In barley a-amylase both Tyr 380 , situated on a remote non-catalytic domain, and Tyr 105 in subsite À6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr 380 and Tyr 105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr 105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants.
European Journal of Biochemistry
Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites 25/26 (Cys95 ! Ala/Thr) and þ1/þ 2 (Met298 ! Ala/ Asn/Ser) as well as in the double mutants, Cys95 ! Ala/ Met298 ! Ala/Asn/Ser. Cys95 ! Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl b-D-maltoheptaoside (Cl-PNPG 7 ), respectively, but fivefold to 20-fold higher K m . The Cys95 ! Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 ! Ala and wild-type. Met298 ! Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k cat and k cat /K m for Cl-PNPG 7 are # 30% and # 10% of wild-type, respectively. The activity of Cys95 ! Ala/Met298 ! Ala/ Asn/Ser is 100-180% towards starch, and the k cat /K m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG 7 , respectively, emphasizing the strong impact of the Cys95 ! Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG 7 and amylose/Cl-PNPG 7 are 2.8-to 270-fold and 1.2-to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites 25 and þ2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites þ1 and þ2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6 000 -maltotriosyl-maltohexaose thus covering subsites 2 1 to þ5, while productive binding of unbranched substrate involves subsites 2 3 to þ3.
Carbohydrate Polymers, 2012
The relationships between starch structure and functionality are important in underpinning the industrial and nutritional utilisation of starches. In this work, the relationships between the biosynthesis, structure, molecular organisation and functionality have been examined using a series of defined genotypes in barley with low (<20%), standard (20-30%), elevated (30-50%) and high (>50%) amylose starches. A range of techniques have been employed to determine starch physical features, higher order structure and functionality. The two genetic mechanisms for generating high amylose contents (down-regulation of branching enzymes and starch synthases, respectively) yielded starches with very different amylopectin structures but similar gelatinisation and viscosity properties driven by reduced granular order and increased amylose content. Principal components analysis (PCA) was used to elucidate the relationships between genotypes and starch molecular structure and functionality. Parameters associated with granule order (PC1) accounted for a large percentage of the variance (57%) and were closely related to amylose content. Parameters associated with amylopectin fine structure accounted for 18% of the variance but were less closely aligned to functionality parameters.