Crystal Structure of TetR Family Repressor AlkX from Dietzia sp. Strain DQ12-45-1b Implicated in Biodegradation of n -Alkanes

Abstract

-Alkanes are ubiquitous in nature and are widely used by microorganisms as carbon sources. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and is used in industrial and environmental applications. The alkane oxidation operon,, in the alkane-degrading strain sp. strain DQ12-45-1b is negatively autoregulated by the TetR family repressor AlkX via a product positive feedback mechanism. To predict the gene regulation mechanism, we determined the 3.1-Å crystal structure of an AlkX homodimer in a non-DNA-bound state. The structure showed traceable long electron density deep inside a hydrophobic cavity of each monomer along the long axis of the helix bundle, and further gas chromatography-mass spectrometry analysis of AlkX revealed that it contained the-derived long-chain fatty acid molecules as a ligand. Moreover, an unusual structural feature of AlkX is an extra helix, α6', forming a lid-like structure with α6 covering the inducer-binding pocket and occupying the space between the two symmetrical DNA-binding motifs in one dimer, indicating a distinct conformational transition mode in modulating DNA binding. Sequence alignment of AlkX homologs from strains showed that the residues involved in DNA and inducer binding are highly conserved, suggesting that the regulation mechanisms of-alkane hydroxylation are possibly a common characteristic of strains. With -alkanes being ubiquitous in nature, many bacteria from terrestrial and aquatic environments have evolved-alkane oxidation functions. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and petroleum-contaminating environment bioremediation. The gene regulation of the most common alkane hydroxylase, AlkB, has been studied widely in Gram-negative bacteria but has been less explored in Gram-positive bacteria. Our previous study showed that the TetR family regulator (TFR) AlkX negatively autoregulated the alkane oxidation operon,, in the Gram-positive strain sp. strain DQ12-45-1b. Although TFRs are one of the most common transcriptional regulator families in bacteria, the TFR involved in-alkane metabolism has been reported only recently. In this study, we determined the crystal structure of AlkX, which implies a distinct DNA/ligand binding mode. Our results shed light upon the regulation mechanism of the common alkane degradation process in nature.

CrgA Protein Represses AlkB2 Monooxygenase and Regulates the Degradation of Medium-to-Long-Chain n-Alkanes in Pseudomonas aeruginosa SJTD-1

“…As AlkB enzymes play main role in the alkane hydroxylation, their regulation mechanisms are concerned. Several regulators have been reported involved in its regulation as activators or repressors, while the regulation of alkane biodegradation remains unclear as the multiple alkane monooxygenase systems and various regulation mode ( Ratajczak et al, 1998 ; Kurth et al, 2008 ; Wang and Shao, 2014 ; Liang et al, 2015 , 2016 , 2017 ). In P. aeruginosa SJTD-1, the global response to alkane environments was observed with the significant change in the expression of many proteins involved in uptake, transportation, carbon metabolism and regulation ( Liu et al, 2015 ; Zhou et al, 2017 ).…”

Extracellular heme recycling and sharing across species by novel mycomembrane vesicles of a Gram-positive bacterium

“…The Dt HtaA was incubated with 10 μM heme at 30 °C for 30 min and then purified by washing [ 44 ]. Simultaneously, a TetR family repressor AlkX from DQ12-45-1b, which is involved in the expression regulation of alkane hydroxylase [ 51 ], was purified and incubated with heme as negative control. The purified protein solutions were scanned from 350 to 500 nm using a microplate reader (Molecular Devices, Sunnyvale, USA).…”

Membrane vesicles from a Dietzia bacterium containing multiple cargoes and their roles in iron delivery

“…DQ12‐45‐1b from oil production water (Wang et al ., 2011). By determination of its capacity for alkane degradation, structural‐based regulation mechanism and production of various biosurfactants (Nie et al ., 2011; Liang et al ., 2016a; Liang et al ., 2017), we showed its potential applications in bioremediation and microbially enhanced oil recovery. In addition, DQ12‐45‐1b was able to adapt to multiple stressors including growth at pH 12 and tolerance of NaCl up to a concentration of 20% (Fang et al ., 2018).…”