Hydrolase CehA and a Novel Two-Component 1-Naphthol Hydroxylase CehC1C2 are Responsible for the Two Initial Steps of Carbaryl Degradation in Rhizobium sp. X9

Abstract

Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH 2 or FADH 2 -dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The K m and K cat /K m values of CehC1 for 1-naphthol were 74.71 ± 16.07 μM and (8.29 ± 2.44) × 10 −4 s −1 •μM −1 , respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.

Conserved Metabolic and Evolutionary Themes in Microbial Degradation of Carbamate Pesticides

“…(A) In order to gain a clear understanding of the structurefunction relationship and the environmental behaviour of various degradation enzymes, it is essential to establish a putative evolutionary path. Although available (Trivedi et al, 2016;Zhou et al, 2020;Jiang et al, 2021), such analyses are very limited for carbamate degradation enzymes and pose a lacuna for future research. (B) The detailed reports characterising the genetic, enzymatic, metabolic and biochemical aspects of carbamate degradation are limited.…”

“…CehC1 functions as an FMNH 2 or FADH 2 -dependent monooxygenase, while CehC2 is a reductase that utilises NADH to reduce FAD or FMN. Compared to the single component 1NH (McbC), the two-component 1NH (CehC1C2) showed significantly lower hydroxylation activity with 1-naphthol, but displayed a broader substrate specificity ( Zhou et al, 2020 ).…”

“…An identical gene arrangement is observed for the cehA encoding CH in Rhodococcus sp. X9 ( Zhou et al, 2020 ). In strain KN65.2, cehA (also known as cfdJ ) is flanked by a truncated istB upstream and a short orf downstream.…”

“…X9 shows 58% similarity with the oxygenase component of two-component 4- nitrophenol 2-monooxygenase from Rhodococcus sp. PN1 and 64% activity on 4-nitrophenol as compared to 1-naphthol ( Zhou et al, 2020 ).…”

Unveiling the CoA mediated salicylate catabolic mechanism in Rhizobium sp. X9

“…Rhizobium sp. X9 is a carbaryl‐degrading strain isolated from pesticide contaminated soil in our previous work and the carbaryl hydrolase gene cehA and a novel 1‐naphthol hydroxylase gene cehC1C2 have been cloned (Zhou et al., 2020). In this study, the complete carbaryl degradation pathway in strain X9 via 1‐naphthol, salicylate and gentisate was predicted based on the bioinformatic analysis, and the salicylate catabolism via a CoA derivative was studied.…”