Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate

ABSTRACT Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.

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The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)38086-4 ◽

1991 ◽

Vol 266 (10) ◽

pp. 6079-6085

Author(s):

T Yano ◽

S Kuramitsu ◽

S Tanase ◽

Y Morino ◽

K Hiromi ◽

...

Keyword(s):

Escherichia Coli ◽

Aspartate Aminotransferase ◽

Catalytic Mechanism

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Site-specific alteration of arginine 376, the unique positively charged amino acid residue in the mid-membrane-spanning regions of the proline carrier of Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37520-8 ◽

1994 ◽

Vol 269 (8) ◽

pp. 5720-5724

Author(s):

I. Yamato ◽

M. Kotani ◽

Y. Oka ◽

Y. Anraku

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Residue ◽

Site Specific ◽

Charged Amino Acid Residue ◽

Specific Alteration ◽

Membrane Spanning ◽

Positively Charged

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

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

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.

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The Unusual Role of Pro in Cu(II) Binding by His2-Cyclopentapeptide

International Journal of Molecular Sciences ◽

10.3390/ijms22126628 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6628

Author(s):

Aleksandra Pieniężna ◽

Aleksandra Kotynia ◽

Justyna Brasuń

Keyword(s):

Amino Acid ◽

Potentiometric Titration ◽

Amino Acid Residue ◽

Strong Impact ◽

Mononuclear Complexes ◽

Study Results ◽

Metal Ratio ◽

Ph Range ◽

L1 And L2

In this paper, we present findings from studying the interaction of copper(II) ions with the His2-cyclopentapeptide and the role of proline used for the purpose of potentiometric titration and UV-Vis, CD and EPR spectroscopic measurements. Experiments of two homodetic peptides differing by one amino acid residue were conducted for a ligand to metal ratio of 1:1 in the pH range 2.5–11.0. The presented studies reveal that peptides form only mononuclear complexes, and the CuH2L complex appears in the system first (for both L1 and L2). Study results show that the presence of Pro influences the structure of formed complexes and their stabilities and has a strong impact on the efficiency of copper(II) coordination.

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Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2007.05.003 ◽

2007 ◽

Vol 1774 (8) ◽

pp. 1029-1035 ◽

Cited By ~ 7

Author(s):

Hiroko Shibata ◽

Haruhiko Kamada ◽

Kyoko Kobayashi-Nishibata ◽

Yasuo Yoshioka ◽

Toshihide Nishibata ◽

...

Keyword(s):

Amino Acid ◽

Tumor Necrosis Factor ◽

Receptor Binding ◽

Amino Acid Residue ◽

Tumor Necrosis ◽

Binding Capacity ◽

Necrosis Factor

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Role of Amino Acid Residue 187 of Poliovirus Polypeptide 2C in Determining the Guanidine Trait

Intervirology ◽

10.1159/000150246 ◽

1992 ◽

Vol 33 (3) ◽

pp. 165-172 ◽

Cited By ~ 3

Author(s):

Robert F. Baltera Jr. ◽

Daniel R. Tershak

Keyword(s):

Amino Acid ◽

Amino Acid Residue

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Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς70

Journal of Bacteriology ◽

10.1128/jb.182.1.221-224.2000 ◽

2000 ◽

Vol 182 (1) ◽

pp. 221-224 ◽

Cited By ~ 6

Author(s):

Christina Wilson Bowers ◽

Andrea McCracken ◽

Alicia J. Dombroski

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Aspartic Acid ◽

Transcription Initiation ◽

Amino Acid Substitutions ◽

Conserved Residues ◽

Acidic Residues

ABSTRACT Amino acid substitutions in Escherichia coliς70 were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

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