scholarly journals Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation

2020 ◽  
Vol 48 (10) ◽  
pp. 5603-5615
Author(s):  
Mihaela-Carmen Unciuleac ◽  
Yehuda Goldgur ◽  
Stewart Shuman
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Active Sites ◽  
Enzyme Structure ◽  
Rna Ligase ◽  
Alanine Scan ◽  
Unexpected Findings ◽  
Metal Cofactors ◽  
Conserved Amino Acids ◽  
Naegleria Gruberi

Abstract Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3′-OH RNA strands in the context of 3′-OH/5′-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

scholarly journals Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii

2015 ◽  
Vol 60 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Shivendra Pratap ◽  
Madhusudhanarao Katiki ◽  
Preet Gill ◽  
Pravindra Kumar ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Crystal Structure ◽  
Acinetobacter Baumannii ◽  
Active Site ◽  
Active Sites ◽  
Multidrug Resistant ◽  
Crystal Structure Analysis ◽  
Content Type ◽  
Class D ◽  
Enzyme Species ◽  
Hydroxyethyl Group

ABSTRACTCarbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistantAcinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL ofA. baumanniifollowing acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45

2005 ◽  
Vol 201 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hyun-Joo Nam ◽  
Florence Poy ◽  
Haruo Saito ◽  
Christin A. Frederick
Keyword(s):  
Crystal Structure ◽  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Active Sites ◽  
Hydrophobic Interactions ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Structural Basis ◽  
Salt Bridges ◽  
Protein Tyrosine

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3ζ immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 “active site” deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen–related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

scholarly journals Crystal Structure of the Catalytic Core of an RNA-Polymerase Ribozyme

Science ◽  
2009 ◽  
Vol 326 (5957) ◽  
pp. 1271-1275 ◽  
Author(s):  
David M. Shechner ◽  
Robert A. Grant ◽  
Sarah C. Bagby ◽  
Yelena Koldobskaya ◽  
Joseph A. Piccirilli ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Active Site ◽  
Rna World ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Catalytic Core ◽  
Rna Ligase ◽  
Catalytic Rate ◽  
A Minor

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

scholarly journals Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

2016 ◽  
Vol 72 (7) ◽  
pp. 523-533 ◽  
Author(s):  
Sergey Lunev ◽  
Soraya S. Bosch ◽  
Fernando de Assis Batista ◽  
Carsten Wrenger ◽  
Matthew R. Groves
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Domain ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Cell Parameters ◽  
High Degree

Thede novopyrimidine-biosynthesis pathway ofPlasmodium falciparumis a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition ofde novopyrimidine biosynthesisviadihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed toN-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure ofPfATC is reported. The space group of thePfATC crystals was determined to be monoclinicP21, with unit-cell parametersa= 87.0,b= 103.8,c= 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presentedPfATC model shares a high degree of homology with the catalytic domain ofEscherichia coliATC. There is as yet no evidence of the existence of a regulatory domain inPfATC. Similarly toE. coliATC,PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.

Catalytic Resonance Theory: Parallel Reaction Pathway Control

2019 ◽  
Author(s):  
M. Alexander Ardagh ◽  
Manish Shetty ◽  
Anatoliy Kuznetsov ◽  
Qi Zhang ◽  
Phillip Christopher ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Control Strategies ◽  
Oscillation Amplitude ◽  
Catalytic Performance ◽  
Parallel Reaction ◽  
Resonance Theory ◽  
Static Conditions

Catalytic enhancement of chemical reactions via heterogeneous materials occurs through stabilization of transition states at designed active sites, but dramatically greater rate acceleration on that same active site is achieved when the surface intermediates oscillate in binding energy. The applied oscillation amplitude and frequency can accelerate reactions orders of magnitude above the catalytic rates of static systems, provided the active site dynamics are tuned to the natural frequencies of the surface chemistry. In this work, differences in the characteristics of parallel reactions are exploited via selective application of active site dynamics (0 < ΔU < 1.0 eV amplitude, 10<sup>-6</sup> < f < 10<sup>4</sup> Hz frequency) to control the extent of competing reactions occurring on the shared catalytic surface. Simulation of multiple parallel reaction systems with broad range of variation in chemical parameters revealed that parallel chemistries are highly tunable in selectivity between either pure product, even when specific products are not selectively produced under static conditions. Two mechanisms leading to dynamic selectivity control were identified: (i) surface thermodynamic control of one product species under strong binding conditions, or (ii) catalytic resonance of the kinetics of one reaction over the other. These dynamic parallel pathway control strategies applied to a host of chemical conditions indicate significant potential for improving the catalytic performance of many important industrial chemical reactions beyond their existing static performance.

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