TY - JOUR
T1 - Crystal structure and functional analysis of the glutaminyl cyclase from Xanthomonas campestris
AU - Huang, Wei Lin
AU - Wang, Yu Ruei
AU - Ko, Tzu Ping
AU - Chia, Cho Yun
AU - Huang, Kai Fa
AU - Wang, Andrew H.J.
N1 - Funding Information:
We thank Dr. Wen-Yih Jeng at the Institute of Biological Chemistry, Academia Sinica (Taipei, Taiwan) for assistance in X-ray data collection. We are grateful to Prof. Shan-Ho Chou at the Institute of Biochemistry of National Chung-Hsing University (Taichung, Taiwan) for kindly providing the genomic DNA of X. campestris. We are also grateful for the access to the synchrotron radiation beamline 13B1 (National Synchrotron Radiation Research Center, Taiwan) and beamline 5A (Photon Factory, Japan). This work was supported by Academia Sinica and Core Facility for Protein Production and X-Ray Structural Analysis of the National Research Program for Genomic Medicine ( NSC97-3112-B-001-035-B4 ).
PY - 2010/8
Y1 - 2010/8
N2 - Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu45) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu89 as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.
AB - Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu45) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu89 as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.
KW - Glutaminyl cyclase
KW - Pyroglutamate
KW - Xanthomonas campestris
KW - β-propeller
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U2 - 10.1016/j.jmb.2010.06.012
DO - 10.1016/j.jmb.2010.06.012
M3 - Article
C2 - 20558177
AN - SCOPUS:77955282532
SN - 0022-2836
VL - 401
SP - 374
EP - 388
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -