Catalytic Mechanism and Product Specificity of Rubisco Large Subunit Methyltransferase:  QM/MM and MD Investigations†

Biochemistry ◽  
2007 ◽  
Vol 46 (18) ◽  
pp. 5505-5514 ◽  
Author(s):  
Xiaodong Zhang ◽  
Thomas C. Bruice
Keyword(s):  
Catalytic Mechanism ◽  
Large Subunit ◽  
Product Specificity

scholarly journals A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

2016 ◽  
Vol 113 (8) ◽  
pp. 2068-2073 ◽  
Author(s):  
Erik W. Debler ◽  
Kanishk Jain ◽  
Rebeccah A. Warmack ◽  
You Feng ◽  
Steven G. Clarke ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Type I ◽  
Histone H4 ◽  
Active Site Residues ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

scholarly journals Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

2015 ◽  
Vol 71 (4) ◽  
pp. 1006-1021 ◽  
Author(s):  
Chien-Chih Lin ◽  
Phimonphan Chuankhayan ◽  
Wen-Ni Chang ◽  
Tseng-Ting Kao ◽  
Hong-Hsiang Guan ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Product Specificity ◽  
Product Release ◽  
Terminal Domain ◽  
Complete Set ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Key Residues

10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86–90, 135–143 and 200–203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

scholarly journals The effects of truncations of the small subunit on m-calpain activity and heterodimer formation

1997 ◽  
Vol 326 (1) ◽  
pp. 31-38 ◽  
Author(s):  
John S. ELCE ◽  
Peter L. DAVIES ◽  
Carol HEGADORN ◽  
Donald H. MAURICE ◽  
J. Simon C. ARTHUR
Keyword(s):  
Catalytic Mechanism ◽  
Large Subunit ◽  
Small Subunit ◽  
Full Length ◽  
Deletion Mutants ◽  
Subunit Interactions ◽  
Calpain Activity ◽  
Specific Activities ◽  
The Stability ◽  
Histidine Tag

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a δ86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-δ86,-δ99, -δ107, and -δ116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-δ125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from δ86, leaving residues 87–245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245–270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys105 → Ser-80k/δ86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca2+-induced dissociation is involved in calpain activation.

scholarly journals Differences in Carbon Isotope Discrimination of Three Variants of D-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Reflect Differences in Their Catalytic Mechanisms

2007 ◽  
Vol 282 (49) ◽  
pp. 36068-36076 ◽  
Author(s):  
Dennis B. McNevin ◽  
Murray R. Badger ◽  
Spencer M. Whitney ◽  
Susanne von Caemmerer ◽  
Guillaume G. B. Tcherkez ◽  
...  
Keyword(s):  
Carbon Isotope ◽  
Rhodospirillum Rubrum ◽  
Catalytic Mechanism ◽  
Higher Plants ◽  
Large Subunit ◽  
Stable Carbon Isotope ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Membrane Inlet Mass Spectrometry ◽  
Carbon Isotope Effect

The carboxylation kinetic (stable carbon) isotope effect was measured for purified d-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO2 as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations (Δ) of 27.4 ± 0.9‰ for wild-type tobacco Rubisco, 22.2 ± 2.1‰ for Rhodospirillum rubrum Rubisco, and 11.2 ± 1.6‰ for a large subunit mutant of tobacco Rubisco in which Leu335 is mutated to valine (L335V). These Δ values are consistent with the photosynthetic discrimination determined for wild-type tobacco and transplastomic tobacco lines that exclusively produce R. rubrum or L335V Rubisco. The Δ values are indicative of the potential evolutionary variability of Δ values for a range of Rubiscos from different species: Form I Rubisco from higher plants; prokaryotic Rubiscos, including Form II; and the L335V mutant. We explore the implications of these Δ values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower Δ value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

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