Combing multiple site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis

ABSTRACT A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.

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The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

Biological Chemistry ◽

10.1515/hsz-2017-0178 ◽

2017 ◽

Vol 399 (1) ◽

pp. 73-77 ◽

Cited By ~ 7

Author(s):

Monika B. Dolinska ◽

Yuri V. Sergeev

Keyword(s):

Oculocutaneous Albinism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Multiple Site ◽

Insect Larvae ◽

Melanin Biosynthesis ◽

Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

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Multiple Site-Directed Mutagenesis In Vitro

In Vitro Mutagenesis Protocols ◽

10.1385/1-59259-194-9:029 ◽

2002 ◽

pp. 29-36 ◽

Cited By ~ 1

Author(s):

Yang-Gyun Kim ◽

Stefan Maas

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Multiple Site

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Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

Biochemical Journal ◽

10.1042/bj20011468 ◽

2002 ◽

Vol 365 (1) ◽

pp. 303-309 ◽

Cited By ~ 20

Author(s):

Wynand B.L. ALKEMA ◽

Antoon K. PRINS ◽

Erik de VRIES ◽

Dick B. JANSSEN

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Catalytic Mechanism ◽

Penicillin Acylase ◽

Precursor Protein ◽

Site Directed Mutagenesis ◽

Penicillin G ◽

Directed Mutagenesis ◽

Wild Type ◽

Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

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Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Biochemistry ◽

10.1021/bi00220a006 ◽

1991 ◽

Vol 30 (6) ◽

pp. 1484-1490 ◽

Cited By ~ 64

Author(s):

David A. Jewell ◽

Chingkuang Tu ◽

Shanthi R. Paranawithana ◽

Susan M. Tanhauser ◽

Philip V. LoGrasso ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Catalytic Properties ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Carbonic Anhydrase Iii ◽

Human Carbonic Anhydrase

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Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714022755 ◽

2014 ◽

Vol 70 (12) ◽

pp. 3253-3265 ◽

Cited By ~ 21

Author(s):

Elena Fernández-Fueyo ◽

Sandra Acebes ◽

Francisco J. Ruiz-Dueñas ◽

María Jesús Martínez ◽

Antonio Romero ◽

...

Keyword(s):

Crystal Structure ◽

Electron Transfer ◽

Manganese Peroxidase ◽

Catalytic Properties ◽

Substrate Oxidation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ceriporiopsis Subvermispora ◽

Ligninolytic Fungi ◽

Access Channel

The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

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Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue

Biochemistry ◽

10.1021/bi00428a015 ◽

1989 ◽

Vol 28 (2) ◽

pp. 510-516 ◽

Cited By ~ 17

Author(s):

A. Martinez del Pozo ◽

M. Merola ◽

H. Ueno ◽

J. M. Manning ◽

K. Tanizawa ◽

...

Keyword(s):

Amino Acid ◽

Spectroscopic Properties ◽

Site Directed Mutagenesis ◽

Tryptophan Residue ◽

Directed Mutagenesis ◽

Multiple Site

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Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8937-8940.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8937-8940 ◽

Cited By ~ 15

Author(s):

Francesca Scaramozzino ◽

Ilona Estruch ◽

Paola Rossolillo ◽

Marco Terreni ◽

Alessandra M. Albertini

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Catalytic Properties ◽

Penicillin G Acylase ◽

Penicillin G ◽

Protein Maturation ◽

Β Chain

ABSTRACT A tag of three lysines alternating with three glycines was added to the C-terminal end of the β chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.

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Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic N-oxides

Catalysts ◽

10.3390/catal9040356 ◽

2019 ◽

Vol 9 (4) ◽

pp. 356 ◽

Cited By ~ 2

Author(s):

Vytautas Petkevičius ◽

Justas Vaitekūnas ◽

Dovydas Vaitkus ◽

Narimantas Čėnas ◽

Rolandas Meškys

Keyword(s):

Organic Chemistry ◽

Escherichia Coli ◽

Active Site ◽

Catalytic Properties ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Work Site ◽

Whole Cells ◽

New Variant

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on – PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML.

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