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

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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Tightening and Untying the Knot in Human Carbonic Anhydrase III

The Journal of Physical Chemistry Letters ◽

10.1021/jz400748b ◽

2013 ◽

Vol 4 (11) ◽

pp. 1829-1833 ◽

Cited By ~ 9

Author(s):

Joachim Dzubiella

Keyword(s):

Carbonic Anhydrase ◽

Carbonic Anhydrase Iii ◽

Human Carbonic Anhydrase

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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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