scholarly journals Identification of Essential Amino Acids in theAzorhizobium caulinodans Fucosyltransferase NodZ

2001 ◽  
Vol 183 (24) ◽  
pp. 7067-7075 ◽  
Author(s):  
Valerie Chazalet ◽  
Kazuyoshi Uehara ◽  
Roberto A. Geremia ◽  
Christelle Breton
Keyword(s):  
Catalytic Domain ◽  
Essential Amino Acids ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Signal Molecules ◽  
Directed Mutagenesis ◽  
Nod Factors ◽  
Peptide Motifs ◽  
Theoretical Approaches ◽  
Complex Glycans

ABSTRACT The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an α1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a “Rossmann-type” nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases.

scholarly journals Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

2003 ◽  
Vol 375 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Takahiro ABE ◽  
Xiaolan LU ◽  
Ying JIANG ◽  
Clark E. BOCCONE ◽  
Shaomin QIAN ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Regulatory Region ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Kinase C ◽  
Acidic Phospholipids ◽  
Ef Hands ◽  
Mutant Forms

Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKα is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKα includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKα also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKα and to determine the enzymic activities of different mutant forms of pig DAGKα in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKα activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKα. Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol. A DAGKα mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation. Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS. These results indicate that Ca2+ and PS stimulate DAGKα via distinct mechanisms.

scholarly journals Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis

2021 ◽  
Author(s):  
Zhengli Bai ◽  
Menglong Xu ◽  
Ying Mei ◽  
Tuo Hu ◽  
Panpan Zhang ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Dissociation Rate ◽  
Antibody Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
High Affinity ◽  
Cdr Grafting ◽  
Slow Dissociation

Abstract Inhibition of Proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. To eliminate the cytotoxic effector functions and mitigate the heterogeneity, the heavy-chain constant region of h5E12-L230G was modified with L234A/L235A/N297G mutations and C-terminal lysine deletion. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD =1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reduced the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to Alirocumab. Our data suggest that h5E12-L230G is a highly potent antibody binding to PCSK9 catalytic domain with slow dissociation rate which may be utilized as a therapeutic candidate for treating hypercholesterolemia and relevant cardiovascular diseases.

scholarly journals Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1783
Author(s):  
Zhengli Bai ◽  
Menglong Xu ◽  
Ying Mei ◽  
Tuo Hu ◽  
Panpan Zhang ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Dissociation Rate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Molecular Docking Study ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
High Affinity ◽  
Cdr Grafting ◽  
Slow Dissociation

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (−54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

scholarly journals Poly(A) polymerase contains multiple functional domains.

1994 ◽  
Vol 14 (5) ◽  
pp. 2946-2957 ◽  
Author(s):  
T Raabe ◽  
K G Murthy ◽  
J L Manley
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Directed Mutagenesis ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

scholarly journals Site-Directed Mutagenesis and Expression of the Soluble Form of the Family IIIa Cellulose Binding Domain from the Cellulosomal Scaffolding Protein of Clostridium cellulovorans

2005 ◽  
Vol 187 (20) ◽  
pp. 7146-7149 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Scaffolding Protein ◽  
Soluble Form ◽  
Directed Mutagenesis ◽  
Cellulose Binding Domain ◽  
Binding Studies ◽  
Clostridium Cellulovorans ◽  
The Family ◽  
Cellulose Binding

ABSTRACT The planar and anchoring residues of the family IIIa cellulose binding domain (CBD) from the cellulosomal scaffolding protein of Clostridium cellulovorans were investigated by site-directed mutagenesis and cellulose binding studies. By fusion with maltose binding protein, the family IIIa recombinant wild-type and mutant CBDs from C. cellulovorans were expressed as soluble forms. Cellulose binding tests of the mutant CBDs indicated that the planar strip residues played a major role in cellulose binding and that the anchoring residues played only a minor role.

scholarly journals Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.

1990 ◽  
Vol 265 (31) ◽  
pp. 19249-19256
Author(s):  
F Simonin ◽  
J Ménissier-de Murcia ◽  
O Poch ◽  
S Muller ◽  
G Gradwohl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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