scholarly journals Metal Binding Motif in the Active Site of the HDV Ribozyme Binds Divalent and Monovalent Ions

Biochemistry ◽  
2011 ◽  
Vol 50 (13) ◽  
pp. 2672-2682 ◽  
Author(s):  
Narayanan Veeraraghavan ◽  
Abir Ganguly ◽  
Jui-Hui Chen ◽  
Philip C. Bevilacqua ◽  
Sharon Hammes-Schiffer ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Binding Motif ◽  
Monovalent Ions ◽  
Hdv Ribozyme ◽  
Metal Binding Motif

scholarly journals Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2961 ◽  
Author(s):  
De-Sheng Ker ◽  
Sze Lei Pang ◽  
Noor Farhan Othman ◽  
Sekar Kumaran ◽  
Ee Fun Tan ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Binding Motif ◽  
Multiple Sequence ◽  
Sesquiterpene Synthase ◽  
Catalytic Active Site ◽  
Soluble Aggregate ◽  
Persicaria Minor ◽  
Metal Binding Motif

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, addition of 15% (v/v) glycerol to the protein purification buffer and removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

scholarly journals Metal ion recognition of the metal-binding motif in hammerhead ribozymes

2003 ◽  
Vol 43 (supplement) ◽  
pp. S28
Author(s):  
Y. Tanaka ◽  
Y. Kasai ◽  
C. Kojima ◽  
K. Yamasaki ◽  
H. Morita ◽  
...  
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Binding Motif ◽  
Hammerhead Ribozymes ◽  
Ion Recognition ◽  
Metal Binding Motif

scholarly journals Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

2004 ◽  
Vol 166 (7) ◽  
pp. 1003-1014 ◽  
Author(s):  
Gideon Lansbergen ◽  
Yulia Komarova ◽  
Mauro Modesti ◽  
Claire Wyman ◽  
Casper C. Hoogenraad ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Binding Motif ◽  
Force Microscopy ◽  
Scanning Force ◽  
Metal Binding Motif

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Efficient Cadmium Bioaccumulation by Displayed Hybrid CS3 Pili: Effect of Heavy Metal Binding Motif Insertion Site on Adsorption Capacity and Selectivity

2015 ◽  
Vol 177 (8) ◽  
pp. 1729-1741 ◽  
Author(s):  
Vajiheh Eskandari ◽  
Bagher Yakhchali ◽  
Mehdi Sadeghi ◽  
Ali Asghar Karkhane ◽  
Houra Ahmadi-Danesh
Keyword(s):  
Heavy Metal ◽  
Adsorption Capacity ◽  
Metal Binding ◽  
Insertion Site ◽  
Binding Motif ◽  
Heavy Metal Binding ◽  
Cadmium Bioaccumulation ◽  
Metal Binding Motif

The metal-binding motif of dipeptidyl peptidase III directly influences the enzyme activity in the copper derivative of dipeptidyl peptidase III

2004 ◽  
Vol 431 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Junzo Hirose ◽  
Hiroshi Kamigakiuchi ◽  
Hiroyuki Iwamoto ◽  
Hideaki Fujii ◽  
Masanori Nakai ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Metal Binding ◽  
Dipeptidyl Peptidase ◽  
Binding Motif ◽  
Metal Binding Motif

scholarly journals Interaction between a metal-binding motif of Hammerhead Ribozyme and divalent cations

2000 ◽  
Vol 40 (supplement) ◽  
pp. S94
Author(s):  
Y. Tanaka ◽  
E.H. Morita ◽  
H. Hayashi ◽  
Y. Kasa ◽  
T. Tanaka ◽  
...  
Keyword(s):  
Metal Binding ◽  
Divalent Cations ◽  
Hammerhead Ribozyme ◽  
Binding Motif ◽  
Metal Binding Motif
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