Improved catalytic properties of a serine hydroxymethyl transferase from Idiomarina loihiensis 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.

Download Full-text

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.

Download Full-text

Site-directed mutagenesis of the Actinomadura R39 DD-peptidase

Biochemical Journal ◽

10.1042/bj3270377 ◽

1997 ◽

Vol 327 (2) ◽

pp. 377-381 ◽

Cited By ~ 7

Author(s):

Guo-Hua ZHAO ◽

Colette DUEZ ◽

Sophie LEPAGE ◽

Christine FORCEILLE ◽

Noureddine RHAZI ◽

...

Keyword(s):

Fusion Proteins ◽

Catalytic Properties ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Structural Elements ◽

Penicillin Binding Protein ◽

Peptide Substrate ◽

Binding Properties ◽

Km Value

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive DD-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ‘SDN loop’ by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.

Download Full-text