In vitro characterization of a nitro-forming oxygenase involved in 3-(trans-2’-aminocyclopropyl)alanine biosynthesis

Mapping Intimacies ◽

10.33774/chemrxiv-2021-zx6rx ◽

2021 ◽

Author(s):

Linlin Pang ◽

Weijing Niu ◽

Yuwei Duan ◽

Liujie Huo ◽

Aiying Li ◽

...

Keyword(s):

Homology Modeling ◽

Heme Oxygenase ◽

Biosynthetic Pathways ◽

Homologous Proteins ◽

Catalytic Center ◽

In Vitro Characterization

In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine ((3-Acp)Ala) and 3-(trans-2’-nitrocyclopropyl)alanine ((3-Ncp)Ala) are originated via two homologous proteins BelK and HrmI, which regioselectively catalyze Nε-nitration of L-lysine. The two enzymes belong to the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily and the catalytic center of BelK is validated by homology modeling and site-directed mutations. Based on the in vitro characterization, the biosynthetic pathways of (3-Acp)Ala and (3-Ncp)Ala are first proposed.

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In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach

International Journal of Molecular Sciences ◽

10.3390/ijms22126243 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6243

Author(s):

Maria Duszyn ◽

Brygida Świeżawska-Boniecka ◽

Aloysius Wong ◽

Krzysztof Jaworski ◽

Adriana Szmidt-Jaworska

Keyword(s):

Brachypodium Distachyon ◽

Bioinformatic Analysis ◽

Catalytic Center ◽

In Vitro Characterization ◽

Plasma Membrane Receptor ◽

Arginine Residues ◽

Catalytic Motif ◽

Conserved Core

In recent years, cyclic guanosine 3′,5′-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules—BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.

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