Identification of Catalytic Bases in the Active Site ofEscherichia coliMethylglyoxal Synthase:  Cloning, Expression, and Functional Characterization of Conserved Aspartic Acid Residues†

Biochemistry ◽  
1998 ◽  
Vol 37 (28) ◽  
pp. 10074-10086 ◽  
Author(s):  
Dana Saadat ◽  
David H. T. Harrison
Keyword(s):  
Aspartic Acid ◽  
Active Site ◽  
Functional Characterization

scholarly journals Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1362
Author(s):  
Cecilia Pozzi ◽  
Stefania Ferrari ◽  
Rosaria Luciani ◽  
Maria Costi ◽  
Stefano Mangani
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Functional Characterization ◽  
Binding Mode ◽  
Anticancer Chemotherapy ◽  
Innovative Strategies ◽  
X Ray Crystallography ◽  
Site Targeting ◽  
New Strategies

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.

scholarly journals Identification and Functional Characterization of an Active-site Lysine in Mevalonate Kinase

1997 ◽  
Vol 272 (9) ◽  
pp. 5741-5746 ◽  
Author(s):  
David Potter ◽  
Jean M. Wojnar ◽  
Chakravarthy Narasimhan ◽  
Henry M. Miziorko
Keyword(s):  
Active Site ◽  
Functional Characterization ◽  
Mevalonate Kinase

scholarly journals Structural and functional characterization of CMP-N-acetylneuraminate synthetase fromVibrio cholerae

2019 ◽  
Vol 75 (6) ◽  
pp. 564-577
Author(s):  
Sucharita Bose ◽  
Debayan Purkait ◽  
Deepthi Joseph ◽  
Vinod Nayak ◽  
Ramaswamy Subramanian
Keyword(s):  
Sialic Acid ◽  
Vibrio Cholerae ◽  
Active Site ◽  
Drug Targets ◽  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Gut Colonization

Several pathogenic bacteria utilize sialic acid, including host-derivedN-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, includingNeisseria meningitidis,Haemophilus influenzae,Pasteurella multocidaandVibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, fromVibrio choleraeare reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure ofV. choleraeCMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

scholarly journals Virus-Encoded Aminoacyl-tRNA Synthetases: Structural and Functional Characterization of Mimivirus TyrRS and MetRS

2007 ◽  
Vol 81 (22) ◽  
pp. 12406-12417 ◽  
Author(s):  
Chantal Abergel ◽  
Joëlle Rudinger-Thirion ◽  
Richard Giegé ◽  
Jean-Michel Claverie
Keyword(s):  
Active Site ◽  
Functional Characterization ◽  
Evolutionary Relationship ◽  
Trna Synthetase ◽  
Dna Viruses ◽  
Trna Synthetases ◽  
Functional Studies ◽  
Aminoacyl Trna Synthetases ◽  
Viral Enzyme

ABSTRACT Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNATyr for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.

Isolation and characterization of a peptide derived from the active site of chicken pepsin

1980 ◽  
Vol 45 (7) ◽  
pp. 2131-2134 ◽  
Author(s):  
Helena Keilová ◽  
Vladimír Kostka ◽  
Miroslav Baudyš
Keyword(s):  
Amino Acid ◽  
Methyl Ester ◽  
Amino Acid Composition ◽  
Aspartic Acid ◽  
Acid Composition ◽  
Active Site ◽  
Its Sequence ◽  
Aspartic Acid Residue ◽  
Isolation And Characterization

A peptide was isolated from chicken pepsin which contains the aspartic acid residue reacting with diazoacetyl-D,L-norleucine methyl ester in the presence of Cu2+ -ions. The peptide is N-terminated with isoleucine and contains (besides isoleucine) valine, aspartic acid, two threonines, serine, and leucine. In concurrent experiments a peptide of the same composition was isolated from the thermolysin digest of chicken pepsin and its sequence determined as Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu. Since both peptides have entirely identical amino acid composition and other characteristics, the sequenced peptide corresponds to the peptide isolated from the active site of the enzyme.

Functional characterization of N-methyl-d-aspartic acid-gated channels in bone cells

Bone ◽  
1999 ◽  
Vol 25 (6) ◽  
pp. 631-637 ◽  
Author(s):  
I Laketić-ljubojević ◽  
L.J Suva ◽  
F.J.M Maathuis ◽  
D Sanders ◽  
T.M Skerry
Keyword(s):  
Aspartic Acid ◽  
Bone Cells ◽  
Functional Characterization
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