Crystallization and preliminary crystallographic analysis of the Escherichia coli tyrosine aminotransferase

1999 ◽  
Vol 55 (8) ◽  
pp. 1474-1477 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Szu-Pei Wu ◽  
Wei-Zen Yang ◽  
Hsin Tsai ◽  
Hanna S. Yuan
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Covalent Bond ◽  
Tyrosine Aminotransferase ◽  
Crystallographic Analysis ◽  
Molecular Replacement ◽  
E Coli ◽  
Open Conformation

Tyrosine aminotransferase catalyzes transamination for both dicarboxylic and aromatic amino-acid substrates. The substrate-free Escherichia coli tyrosine aminotransferase (eTAT) bound with the cofactor pyridoxal 5′-phosphate (PLP) was crystallized in the trigonal space group P32. A low-resolution crystal structure of eTAT was determined by molecular-replacement methods. The overall folding of eTAT resembles that of the aspartate aminotransferases, with the two identical subunits forming a dimer in which each monomer binds a PLP molecule via a covalent bond linked to the ∊-NH2 group of Lys258. Comparison of the structure of eTAT with those of the open, half-open or closed form of chicken or E. coli aspartate aminotransferases shows the eTAT structure to be in the open conformation.

scholarly journals Crystal structure of the aromatic-amino-acid aminotransferase fromStreptococcus mutans

2019 ◽  
Vol 75 (2) ◽  
pp. 141-146
Author(s):  
Xuzhen Cong ◽  
Xiaolu Li ◽  
Shentao Li
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Streptococcus Mutans ◽  
Aromatic Amino Acid ◽  
Aromatic Amino Acids ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Aromatic Amino Acid Aminotransferase

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase fromStreptococcus mutans(SmAroAT) was recombinantly expressed inEscherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure ofSmAroAT was solved at 2.2 Å resolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Crystal structure of Aspergillus fumigatus AroH , an aromatic amino acid aminotransferase

2021 ◽  
Author(s):  
Sharon Spizzichino ◽  
Gioena Pampalone ◽  
Mirco Dindo ◽  
Agostino Bruno ◽  
Luigina Romani ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Aspergillus Fumigatus ◽  
Aromatic Amino Acid ◽  
Aromatic Amino Acid Aminotransferase

scholarly journals Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins

2004 ◽  
Vol 48 (10) ◽  
pp. 3996-4001 ◽  
Author(s):  
Yolanda Sáenz ◽  
Laura Briñas ◽  
Elena Domínguez ◽  
Joaquim Ruiz ◽  
Myriam Zarazaga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Amino Acid ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Mechanisms Of Resistance ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Class 1 ◽  
Human Animal

ABSTRACT Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.

scholarly journals d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

1992 ◽  
Vol 282 (3) ◽  
pp. 747-752 ◽  
Author(s):  
O A M al-Bar ◽  
C D O'Connor ◽  
I G Giles ◽  
M Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Phosphinic Acid ◽  
Mature Protein ◽  
Bamhi Fragment ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

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