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Biotechnol Lett (2009) 31:14451449 DOI 10.1007/s10529-009-0013-6
ORIGINAL RESEARCH PAPER
Mutation in the RGD motif decreases the esterase activity of Xcc_est
Jianjun Wang Yanping Cao Guojun Zheng
Received: 6 March 2009 / Revised: 14 April 2009 / Accepted: 28 April 2009 / Published online: 21 May 2009 Springer Science+Business Media B.V. 2009
Abstract Five truncated constructs of Xcc_est GDSL esterase from Xanthomonas campestris were heterologously expressed and puried. The truncated constructs with a RGD motif had higher specic activities than those without the motif. The specic activity of wild-type Xcc_est was 32.5 2.7 U/mg, while the RGD mutant was 12.5 4.9 U/mg. Moreover, we expressed mature forms of the Xcc_est protein and the RGD mutant as inclusion bodies and, after refolding, there was no signicant difference between the two constructs in specic activity. These results suggest that the RGD motif affects the esterase-domain folding in vivo during the translocation process.
Keywords GDSL esterase Refolding
RGD motif Xanthomonas campestris
Introduction
Most GDSL esterases (Arpigny and Jaeger 1999) consist of two domains: one is a surface-exposed N-terminal passenger domain (or a-domain) that harbors an active site serine in a GDSL motif and other residues in the catalytic site; and the other is a C-terminal b-domain located in the outer membrane (Henderson et al. 1998). A previous study cloned a GDSL esterase gene (Xv_EstE) from Xanthomonas vesicatoria DSM 50861 and over-expressed it in Escherichia coli (Talker-Huiber et al. 2003). Recently, our laboratory also cloned and expressed a highly similar esterase Xcc_est from Xanthomonas campestris pv. campestris (Xcc) 8004 (Wang et al. 2009). Both esterase genes have a RGD (Arg-Gly-Asp) tri-peptide motif in the linker region between the N-terminal domain and the C-terminal b-barrel domain. Here, we show that the RGD motif can affect N-terminal esterase function, suggesting that the motif is involved in the folding of Xcc_est in vivo during translocation.
Materials and methods
Bacterial strains, plasmids and culture media
E. coli DH5a was used for cloning and E. coli BL21 (DE3) was used as the host for expression experiments. pET30a(?) was used for standard cloning
Jianjun Wang and Yanping Cao are contributed equally to this work.
J. WangState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, Peoples Republic of China
Y. Cao...