Residues His172 and Lys238 are Essential for the Catalytic Activity of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain L-1

Abstract

Maleylacetate reductase (PcpE), the last enzyme in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum L-1, catalyzes two consecutive reductive reactions, reductive dehalogenation of 2-chloromaleylacetate (2-CMA) to maleylacetate (MA) and subsequent reduction of MA to 3-oxoadipate (3-OXO). In each reaction, one molecule of NADH is consumed. To better understand its catalytic function, we undertook a structural model-based site-directed mutagenesis and steady-state kinetics study of PcpE. Our results showed that the putative catalytic site of PcpE is located in a positively charged solvent channel at the interface of the two domains and the binding of 2-CMA/MA involves seven basic amino acids, His172, His236, His237, His241 and His251, Lys140 and Lys238. Mutagenesis studies showed that His172 and Lys238 are essential for the catalytic activity of PcpE. However, the mutation of His236 to an alanine can increase the catalytic efficiency (k cat/K m) of PcpE by more than 2-fold, implying that PcpE is still in an early stage of molecular evolution. Similar to tetrachlorobenzoquinone reductase (PcpD), PcpE is also inhibited by pentachlorophenol in a concentration-dependent manner. Furthermore, our studies showed that PcpE exhibits an extremely low but detectable level of alcohol dehalogenase activity toward ethanol and supports the notion that it is evolved from an iron-containing alcohol dehydrogenase.

Soil and foliar application of rock dust as natural control agent for two-spotted spider mites on tomato plants

“…The action of silicon in promoting plant resistance has not been well characterized, but evidence for ex planta and in planta processes are given indicating multiple combined effects rather than one single effect 10 , and silicon has been proposed to act either directly or indirectly on insect herbivores 37 . Several studies have been performed to investigate the effects of Si as a nutrient capable of providing some measures of defense in plants against herbivores and pathogens 14 , 33 , 38 – 42 . It is generally recognized that silicon is able to affect folivorous, phloem-feeding, and some xylem-feeding pests, and action is not only linked to leaf mechanical properties 41 , 42 , but also to indirect induced chemical defenses putatively mediated by soluble Si 7 , 8 , 37 , 43 .…”

“…Several studies have been performed to investigate the effects of Si as a nutrient capable of providing some measures of defense in plants against herbivores and pathogens 14 , 33 , 38 – 42 . It is generally recognized that silicon is able to affect folivorous, phloem-feeding, and some xylem-feeding pests, and action is not only linked to leaf mechanical properties 41 , 42 , but also to indirect induced chemical defenses putatively mediated by soluble Si 7 , 8 , 37 , 43 . The morphological changes observed at the tissue level after exposing plants to a silicon source may be linked to an increase in lignified cells 44 and to epidermal thickening (Fig.…”

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system

“…The notion that microbiota is a critical node in the gut–brain axis is also supported by the observation that colitis, which depends on the gut microbiota, shows significant comorbidities with depression 19 . Finally, probiotic intervention has been shown to influence emotional behavior in animal models of depression 20 and improve mood in depressive patients 21 . However, the molecular mechanisms linking intestinal microbiota and mood disorders remain largely unknown, partly due to the lack of experimental models.…”

Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease

“…It has an 8,000-fold higher affinity to the enzyme than its substrates, i.e., ethanol [23,33] and is reported to have negligible side effects [59]. Its affinity for the bacterial versions of the enzyme is not known, but it has demonstrated efficacy in their blocking [22,60]. In fomepizole-treated Spn, the NAD(H) pool and intracellular ATP concentration were accordingly reduced, and erythromycin susceptibility was increased by more than 5-fold in vivo.…”