The amine-donor substrate specificity of tissue-type transglutaminase. Influence of amino acid residues flanking the amine-donor lysine residue

ABSTRACT Lysine decarboxylase (LDC; EC 4.1.1.18 ) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both l-lysine andl-ornithine with similar Km andVmax values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17 ), including the mouse,Saccharomyces cerevisiae, Neurospora crassa,Trypanosoma brucei, and Caenorhabditis elegansenzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved inS. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward bothl-lysine and l-ornithine with a substrate specificity ratio of 0.83 (defined as thek cat/Km ratio obtained with l-ornithine relative to that obtained withl-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.

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Amino Acid Residues Involved in the Substrate Specificity of TauT/SLC6A6 for Taurine and γ-Aminobutyric Acid

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b13-00991 ◽

2014 ◽

Vol 37 (5) ◽

pp. 817-825 ◽

Cited By ~ 6

Author(s):

Tohru Yahara ◽

Masanori Tachikawa ◽

Shin-ichi Akanuma ◽

Yoshiyuki Kubo ◽

Ken-ichi Hosoya

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Aminobutyric Acid ◽

Amino Acid Residues

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Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p inSaccharomyces cerevisiae

Yeast ◽

10.1002/yea.792 ◽

2001 ◽

Vol 18 (15) ◽

pp. 1429-1440 ◽

Cited By ~ 15

Author(s):

Birgitte Regenberg ◽

Morten C. Kielland-Brandt

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Amino Acid Residues ◽

Amino Acid Permeases

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Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

The Journal of Lipid Research ◽

10.1194/jlr.m064121 ◽

2015 ◽

Vol 57 (1) ◽

pp. 89-99 ◽

Cited By ~ 16

Author(s):

Kenshi Watanabe ◽

Makoto Ohno ◽

Masahiro Taguchi ◽

Seiji Kawamoto ◽

Kazuhisa Ono ◽

...

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Substrate Specificity ◽

Amino Acid Residues ◽

Fatty Acid Desaturases ◽

Front End ◽

Membrane Bound

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Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases ofNeisseria meningitidis

Molecular Microbiology ◽

10.1111/j.1365-2958.2008.06580.x ◽

2009 ◽

Vol 71 (4) ◽

pp. 960-971 ◽

Cited By ~ 28

Author(s):

Heike Claus ◽

Katharina Stummeyer ◽

Julia Batzilla ◽

Martina Mühlenhoff ◽

Ulrich Vogel

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Donor Substrate

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The N-Terminal Amino Acid Residues Ser764 and Lys773 of the D' Domain of Von Willebrand Factor Contribute to Factor VIII Binding

Blood ◽

10.1182/blood.v118.21.2248.2248 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2248-2248

Author(s):

Lydia Castro-Núñez ◽

Esther Bloem ◽

Carmen van der Zwaan ◽

Koen Mertens ◽

Alexander B Meijer

Keyword(s):

Amino Acid ◽

Chemical Modification ◽

Factor Viii ◽

Von Willebrand Factor ◽

Terminal Region ◽

Lysine Residue ◽

Amino Acid Residues ◽

N Terminus ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 2248 Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D'-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D' domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D' domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D' domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D' domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. Disclosures: No relevant conflicts of interest to declare.

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