Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Fushinobu, Shinya; Itō, Kiyoshi; Konno, Masae; Wakagi, Takayoshi; Matsuzawa, Hiroshi

doi:10.1093/protein/11.12.1121

Cited by 138 publications

(111 citation statements)

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“…With one exception, all members of GH54 are from eukaryotic (fungal) sources. A. kawachii is an industrially important fungus for brewing shochu (traditional Japanese liquor), 38) producing various hemicellulases, including acidophilic xylanase, 39) feruloyl esterase, 40) and two different α L arabinofuranosidases, including AkAbf54. 41) These two arabinofuranosidases also support the action of feruloyl esterase to release ferulic acid, which is converted to the flavor compounds in sho- chu brewing.…”

Section: Gh42: a Possible Link Between Retaining And Invert-mentioning

confidence: 99%

New Structural Insights on Carbohydrate-active Enzymes

Fushinobu¹,

Hidaka²,

Miyanaga³

et al. 2007

J. Appl. Glycosci.

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Polysaccharides show a huge degree of diversity owing to their variety of sugar components (aldoses, ketoses and their stereoisomers), the variety of glycosidic bonds (e.g., α 1,4 , α 1,6 , β 1,4 , β 1,3 , etc.), different degrees of polymerization, and different degrees of branching. Therefore, they can be involved in many types of biological function with distinct physicochemical characteristics. It is especially intriguing that α and β glucans, such as amylose (starch) and cellulose, are built from the same glucose units, differing only in the anomeric configurations of glycosidic bonds. Enzymes that hydrolyze these glucans, amylases and cellulases, are the two largest groups of glycoside hydrolases, and have been studied for a long time. A framework to discuss a whole variety of glycoside hydrolases with their three dimensional structural aspects has been established within the last 15 years. In the late 1980s, Henrissat et al. began classification of the amino acid sequences of enzymes, focusing on cellulases and hemicellulases. 1) At the earliest stage of their classification, the α amylase family was treated as belonging to a separate system.2) In 1991, the naming system of the families changed from alphabetical to numerical, and the system expanded to 35 "glycosyl hydrolase" families, incorporating three well known amylase families, GH13 (retaining α amylase family), GH14 (inverting β amylase family), and GH15 (inverting glucoamylase family), as well as many other enzymes.3) All enzymes cleaving, modifying, and creating glycosidic bonds, and modules binding to carbohydrates have been incorporated into the CAZy database (http: www.cazy.org CAZY ). Glycoside hydrolase (GH) families now include enzymes that hydrolyze the glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety. At present, there are 101 families in the GH class ( Fig. 1; note, five have been deleted or reassigned to other classes), and it will continue to grow.The first 3 D structure of a glycoside hydrolase to be determined was that of hen egg white lysozyme (classified into GH22) in the mid 1960s.4) Even in the early 1990s, however, only a limited number of GH enzyme structures were available. Henrissat and colleagues took amino acid sequence information as the primary basis for classification, but they also used hydrophobic cluster analysis from the earliest stage, trying to incorporate as much 3 D structure based information as possible.5) The significant advances in structural studies of GH enzymes over the last decade have indicated that these enzymes show a vast array of tertiary scaffolds, described as "a marketplace of different structures". 6)Our group has been trying to solve crystal structures of useful enzymes for use in bioindustry. Two of these were the first crystal structures within the GH42 and GH57 families, 7,8) and we then began to focus on CAZy enzymes belonging to structurally unknown families. We have since solved two more GH families, and provided the first crystal s...

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Section: Gh42: a Possible Link Between Retaining And Invert-mentioning

confidence: 99%

New Structural Insights on Carbohydrate-active Enzymes

Fushinobu¹,

Hidaka²,

Miyanaga³

et al. 2007

J. Appl. Glycosci.

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“…A. kawachii IFO4308 produces various hemicellulases including an acidophilic xylanase (12) and two different ␣-L-arabinofuranosidases, arabinofuranosidase A (AkAbfA) and arabinofuranosidase B (AkAbfB) (13). AkAbfA and AkAbfB are assigned to GH51 and GH54, respectively.…”

mentioning

confidence: 99%

Crystal Structure of a Family 54 α-l-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose

Miyanaga

Koseki

Matsuzawa

et al. 2004

Journal of Biological Chemistry

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As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 ␣-L-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a ␤-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a ␤-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/ base residues were determined to be Glu 221 and Asp 297 , respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

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“…This is a different strategy to that observed in the acid-stable (but not thermostable) xylanase from Aspergillus kawachii in which many acidic residues are exposed (although even that protein is expected to be positively charged at the pH optimum of 2). 31 The preponderance of basic residues would seem to have the advantage that their high pK a values are outside the pH range for optimum activity (pH 2.5 -7), which would effectively protect the protein from destabilizing changes in surface charge if the pH in the environment varies. This is in fact the case with more "normal" proteins: the pK a values of the vast majority of their surface groups (3 -5 for acidic residues and 9-11 for basic residues) are outside the usual pH range of their environment.…”

mentioning

confidence: 99%

X-ray Structures of the Maltose–Maltodextrin-binding Protein of the Thermoacidophilic Bacterium Alicyclobacillus acidocaldarius Provide Insight into Acid Stability of Proteins

Schäfer

Magnusson

Scheffel

et al. 2004

Journal of Molecular Biology

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Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo-and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of b-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.

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Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Cited by 138 publications

References 1 publication

New Structural Insights on Carbohydrate-active Enzymes

New Structural Insights on Carbohydrate-active Enzymes

Crystal Structure of a Family 54 α-l-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose

X-ray Structures of the Maltose–Maltodextrin-binding Protein of the Thermoacidophilic Bacterium Alicyclobacillus acidocaldarius Provide Insight into Acid Stability of Proteins

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