Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism

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.

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CHEMICAL FUNCTIONS OF AMINO ACID RESIDUES IN THE ACTIVE SITE OF PARAHYDROXYBENZOATE HYDROXYLASE AS STUDIED BY SITE DIRECTED MUTAGENESIS AND BIOPHYSICAL TECHNIQUES

Flavins and Flavoproteins 1990 ◽

10.1515/9783110855425-043 ◽

1991 ◽

pp. 219-230

Author(s):

Barrie Entsch ◽

Bruce A. Palfey ◽

David P. Ballou ◽

Vincent Massey

Keyword(s):

Amino Acid ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Biophysical Techniques

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Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/s0005-2728(97)00085-6 ◽

1998 ◽

Vol 1363 (1) ◽

pp. 85-93 ◽

Cited By ~ 11

Author(s):

Stefan Schmitz ◽

Marta Martı́nez-Júlvez ◽

Carlos Gómez-Moreno ◽

H Böhme

Keyword(s):

Amino Acid ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Positively Charged

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

Biochemistry ◽

10.1021/bi5009112 ◽

2014 ◽

Vol 53 (44) ◽

pp. 6924-6933 ◽

Cited By ~ 5

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

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Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

Biochemical Journal ◽

10.1042/bj2880117 ◽

1992 ◽

Vol 288 (1) ◽

pp. 117-121 ◽

Cited By ~ 48

Author(s):

E P Ko ◽

H Akatsuka ◽

H Moriyama ◽

A Shinmyo ◽

Y Hata ◽

...

Keyword(s):

Amino Acids ◽

Reaction Mechanism ◽

Amino Acid ◽

Bacillus Pumilus ◽

Specific Activity ◽

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Sequence Similarity ◽

Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

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Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4)

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03151-z ◽

2019 ◽

Vol 76 (24) ◽

pp. 4905-4921 ◽

Cited By ~ 3

Author(s):

Yuya Futagi ◽

Masaki Kobayashi ◽

Katsuya Narumi ◽

Ayako Furugen ◽

Ken Iseki

Keyword(s):

Amino Acid ◽

Homology Modeling ◽

Site Directed Mutagenesis ◽

Monocarboxylate Transporters ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Selection Mechanism ◽

Substrate Selection ◽

Identify Amino Acid

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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

Biochemical Journal ◽

10.1042/bj3230415 ◽

1997 ◽

Vol 323 (2) ◽

pp. 415-419 ◽

Cited By ~ 94

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.

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Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine

Biochimie ◽

10.1016/j.biochi.2017.05.013 ◽

2017 ◽

Vol 139 ◽

pp. 125-136 ◽

Cited By ~ 6

Author(s):

Anna G. Mikhailova ◽

Tatiana V. Rakitina ◽

Vladimir I. Timofeev ◽

David M. Karlinsky ◽

Dmitry A. Korzhenevskiy ◽

...

Keyword(s):

Amino Acid ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Serratia Proteamaculans ◽

Oligopeptidase B

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Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1809 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1809-1813 ◽

Cited By ~ 19

Author(s):

R G Chipperfield ◽

S S Jones ◽

K M Lo ◽

R A Weinberg

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Normal Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Transforming Activity ◽

Ras P21 ◽

Ras Oncogenes ◽

Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

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