Loops und Tunnel: unterschätzte Elemente in Enzymen

Heinemann, Peter M.; Rapp, Lea R.; Hauer, Bernhard

doi:10.1007/s12268-020-1394-2

Cited by 1 publication

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“…A similar loop, also probably functioning as an gatekeeper, has been reported in the cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 (Dong et al 2005), which shares a sequence identity of 33.80-37.44 % to these α-subunits. Molecular tunnels, and their flexible loop-sites, in general play important roles for enzyme engineering ( Kreß et al, 2018;Heinemann et al, 2020;Kokkonen et al, 2019). It was shown that they influence not only the specificity and selectivity of an enzyme by controlling the substrate entrance and product release.…”

Section: Discussionmentioning

confidence: 99%

Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli

Hunold

Escobedo‐Hinojosa

Potoudis

et al. 2021

Appl Microbiol Biotechnol

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Phenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile. Key points • Phenylobacterium immobile E DSM 1986 harbors the only described pyrazon oxygenase (PPO). • We elucidated the genes encoding for all PPO components. • Heterologous expression of PPO enabled pyrazon dihydroxylation in E. coli JW5510.

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