title: A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.
abstract: Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Li
authors.forename: Xianli
authors.initials: X
authors.lastname: Xue
authors.forename: Heng
authors.initials: H
authors.lastname: Zhao
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000818:Animals; D002151:Calorimetry; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029964:Fagus / Q000737:chemistry; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011485:Protein Binding; D011487:Protein Conformation; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012417:Rumen / Q000382:microbiology; D017422:Sequence Analysis, DNA; D012756:Sheep; D013696:Temperature; D014908:Triticum / Q000737:chemistry; D014990:Xylans / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D014990:Xylans; D014995:Xylosidases