scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhi-Zhong Song ◽  
Bin Peng ◽  
Zi-Xia Gu ◽  
Mei-Ling Tang ◽  
Bei Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Peach Fruit ◽  
Alcohol Acyltransferase

AbstractThe aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV

Biochemistry ◽  
1992 ◽  
Vol 31 (20) ◽  
pp. 4808-4815 ◽  
Author(s):  
Ursel Selent ◽  
Thomas Rueter ◽  
Eleonore Koehler ◽  
Michaela Liedtke ◽  
Vera Thielking ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Endonuclease ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Identify Amino Acid ◽  
A Site ◽  
Mutagenesis Study

Identification of Amino Acid Residues Underlying the Antiport Mechanism of the Mitochondrial Carnitine/Acylcarnitine Carrier by Site-Directed Mutagenesis and Chemical Labeling

Biochemistry ◽  
2014 ◽  
Vol 53 (44) ◽  
pp. 6924-6933 ◽  
Author(s):  
Nicola Giangregorio ◽  
Lara Console ◽  
Annamaria Tonazzi ◽  
Ferdinando Palmieri ◽  
Cesare Indiveri
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Chemical Labeling

scholarly journals Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

1997 ◽  
Vol 109 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Laurent Schild ◽  
Estelle Schneeberger ◽  
Ivan Gautschi ◽  
Dmitri Firsov
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Site Directed Mutagenesis ◽  
Ion Permeation ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Na Ions ◽  
Membrane Spanning ◽  
A Site ◽  
Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a Ki for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

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