Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

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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Study of substrate specificity of human aromatase by site directed mutagenesis

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2002.02779.x ◽

2002 ◽

Vol 269 (5) ◽

pp. 1393-1405 ◽

Cited By ~ 17

Author(s):

P. Auvray ◽

C. Nativelle ◽

R. Bureau ◽

P. Dallemagne ◽

G.-E. Séralini ◽

...

Keyword(s):

Substrate Specificity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Human Aromatase

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Site-directed mutagenesis of AMP-binding residues in adenylate kinase Alteration of substrate specificity

FEBS Letters ◽

10.1016/0014-5793(93)81687-u ◽

1993 ◽

Vol 334 (1) ◽

pp. 86-88 ◽

Cited By ~ 12

Author(s):

Toshihide Okajima ◽

Katsuyuki Tanizawa ◽

Toshio Fukui

Keyword(s):

Substrate Specificity ◽

Adenylate Kinase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Binding Residues

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Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01628-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Efstratios Nikolaivits ◽

Maria Dimarogona ◽

Ioanna Karagiannaki ◽

Angelina Chalima ◽

Ayelet Fishman ◽

...

Keyword(s):

Substrate Specificity ◽

Fruits And Vegetables ◽

Homology Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Encoding ◽

Content Type ◽

Polyphenol Oxidases ◽

Increased Activity ◽

Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

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