Catalytic Mechanism and Product Specificity of Oxidosqualene-Lanosterol Cyclase: A QM/MM Study

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.

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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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715002928 ◽

2015 ◽

Vol 71 (4) ◽

pp. 1006-1021 ◽

Cited By ~ 3

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.

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

Biochemistry ◽

10.1021/bi700119p ◽

2007 ◽

Vol 46 (18) ◽

pp. 5505-5514 ◽

Cited By ~ 15

Author(s):

Xiaodong Zhang ◽

Thomas C. Bruice

Keyword(s):

Catalytic Mechanism ◽

Large Subunit ◽

Product Specificity

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

10.26434/chemrxiv.7985618.v1 ◽

2019 ◽

Author(s):

Adrian Roitberg ◽

Pancham Lal Gupta

Keyword(s):

Transfer Reaction ◽

Catalytic Mechanism ◽

Ph Dependence ◽

Ph Effects ◽

Dependent Manner ◽

E Coli ◽

Gar Tfase ◽

Activity Curve ◽

Ph Dependent ◽

Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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Arylboronic Acids in Self-condensation of Glucosamines Forming Deoxyfructosazine, Catalysts or Reagents?

10.26434/chemrxiv.7528376 ◽

2019 ◽

Author(s):

Meifeng Wang ◽

Gan Zhu ◽

Yiqun Li ◽

Liuqun Gu

Keyword(s):

Molecular Recognition ◽

Boric Acid ◽

Boronic Acid ◽

Catalytic Mechanism ◽

Organic Transformations ◽

Food Industries ◽

Arylboronic Acids ◽

Excess Amount ◽

Phenyl Boronic Acid ◽

Boron Acid

Arylboronic acids were widely used as efficient catalysts in direct amide formation and other organic transformations. Surprisingly, reports on their use as catalysts in carbohydrates synthesis are very rare even though boron acid-diol complexation was extensively investigated in molecular recognition for saccharides and so on. Here we developed an efficient arylboronic acids catalyzed dimerization of glucosamines forming deoxyfructosazine which is important compound in pharmaceutical and food industries, against a commonly held belief that excess amount of phenyl boronic acid (or boric acid) is a must. A catalytic mechanism was also proposed and arylboronic acids instead of their boronates was identified as catalysts.

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