Importance of Interactions of the .ALPHA.-Helices in the Catalytic Domain N- and C-Terminals of the Family 10 Xylanase from Streptomyces olivaceoviridis E-86 to the Stability of the Enzyme

Abstract

The plant cell wall consists mainly of a complex mixture of polysaccharides such as cellulose, hemicellulose and pectin. 1) Xylan is the major component of the hemicelluloses found in plant cell walls. The backbone of xylan is formed by β 1,4 linked D xylopyranose units to which several side groups such as α 1,2 linked 4 O methyl D glucuronic acid and α 1,3 linked L arabinofuranose are attached. 2)β Xylanase (EC 3.2.1.8) randomly hydrolyzes β 1,4 glycosidic linkages within the xylan backbone to yield short chain xylooligosaccharides of varying length. Xylanases have many commercial uses, such as in the paper manufacturing, animal feed, bread making, juice and wine industries, and xylitol and xylooligosaccharide production.3)Thermostability is an important property of industrially significant enzymes. Understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysis. However, the term thermostability does not refer to a well defined physico chemical property of a protein, but is generally used to indicate the ability of some proteins to function at elevated temperatures and to withstand them for some time. Such an ability implies that the protein is able to remain folded in its functional native state under such conditions. Because many other factors besides temperature affect protein stability, thermostability at best can only be loosely defined. A number of previous studies have shown that various factors can increase protein thermostability, such as an efficient packing of the hydrophobic core, 4 6) electrostatic interactions such as salt bridges and hydrogen bonds, 5,7,8) and the stabilization of helix dipoles. 9)Streptomyces olivaceoviridis E 86 produces both family 10 and 11 xylanases. 10,11) The family 10 xylanase (SoXyn10A) has been well characterized.10,12 22) SoXyn10A consists of two domains, a catalytic domain (belonging to GH family 10) and a carbohydrate binding module (CBM) (belonging to CBM family 13), both of which are connected by a Gly Pro rich linker region.10) The deletion of the CBM of SoXyn10A resulted in about a 20 C decrease in the optimum temperature of SoXyn10A from Streptomyces olivaceoviridis E 86 after removal of the C terminal CBM. Abstract: The intact crystal structure of family 10 xylanase (SoXyn10A) from Streptomyces olivaceoviridis indicates that the catalytic domain of SoXyn10A consists of nine α-helices (α0 8) and eight β-sheets (β1 8). Interaction in the α-helices of N-terminal (α0) and C-terminal (α8) of catalytic domain of SoXyn10A by 3 hydrogen bonds and 8 hydrophobic interactions is observed and predicted to playing an important role for the stability of the molecule. Therefore, the importance for the stability and folding of SoXyn10A were examined by using C-terminal truncated mutants of SoXyn10A. The thermostability was gradually decreased when the C-terminal was shortened; however, the enzyme activities were not influenced by the length of the C-terminal. The investigation of the stability using guanidine ...

Functional Characterization of the GH10 and GH11 Xylanases from <i>Streptomyces olivaceoviridis</i> E-86 Provide Insights into the Advantage of GH11 Xylanase in Catalyzing Biomass Degradation

“…One of the xylanases produced by S. olivaceoviridis E-86 is SoXyn10A (formerly FXYN), which is one of the most well-characterized xylanases. 15) 16) 17) The following functional properties of SoXyn10A have been studied in detail: sugar-binding structures with various kinds of xylooligosacchrides, 18) 19) mechanism of enzyme catalysis, 20) 21) 22) topology of the substrate binding cleft, 23) 24) protein engineering 25) 26) 27) and production of xylooligosaccharides. 28) 29) 30) 31) 32) In contrast, the other xylanase produced by S. olivaceoviridis E-86 designated as SoXyn11B (formerly GXYN) has not been well-characterized.…”

Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant

“… 6) For characterization, a construct, CD (303), consisting of only the catalytic module (residues 1–303) was used, while the full-length enzyme (residues 1–436) was used for crystallization. 4) To facilitate purification, His 6 -tag was added to the C-terminus of the expressed CD (303). The His 6 -tagged enzymes were purified by Ni-nitrilotriacetic acid agarose (Qiagen GmbH, Hilden, Germany) affinity chromatography as described previously.…”

“… 1) 2) SoXyn10A is one of the most widely investigated glycoside hydrolases for studies on reaction mechanism and substrate specificity. 3) 4) 5) 6) 7) 8) 9) In most cases, the retaining enzymes possess glutamic or aspartic acids as acid/base catalyst and nucleophile. 10) 11) The catalytic domain of SoXyn10A forms a (β/α) 8 TIM-barrel structure belonging to clan GH-A, and contains an acid/base catalyst (Glu128) and nucleophile (Glu236) at the active site cleft.…”

Structure and Function of Carbohydrate-Binding Module Families 13 and 42 of Glycoside Hydrolases, Comprising a β-Trefoil Fold

“…In addition to our work on SoCBM13, we have studied the function and structure of the catalytic module by various approaches including mutagenesis, module shuffling, and structural analysis. [62][63][64][65][66] …”