期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 404, 期 1, 页码 100-111出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.09.013
关键词
active site; beta-elimination; polysaccharide lyase family 4; pectin degradation
资金
- Danish Agency for Science, Technology and Innovation
- European Union
- Danish Council for Independent Research
We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the -3/+3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the beta-elimination on [-,2)-alpha-L-rhamno-(1,4)-alpha-D-galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis. (C) 2010 Elsevier Ltd. All rights reserved.
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