Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria

Abstract

Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD⁺. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg^-1. Compared with that, both StyDs showed only activities less than 0.2 U mg^-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg^-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

Keywords: Esterase activity; Evolutionary ancestry; Maximum reaction rate; Michaelis constant; NAD+; Oxidoreductase; Turnover number.

Fig. 1

Comparison of the sty genes organization of Rhodococcus opacus 1CP (CP009112) [39], Pseudomonas putida S12 (CP009975) [2], Gordonia rubripertincta CWB2 (CP022580) [36], and Sphingopyxis fribergensis Kp5.2 (CP009122) [38]. The genes involved in the upper styrene metabolism are styA and styB (encode the SMO), styC (encodes the SOI), styD, and feaB (encode the PAD), respectively. The gene styC2 encodes a protein similar to StyC, and the protein products of styS and styR are a sensor and a regulator proteins [38]

Fig. 2

Michaelis-Menten plots of (a) FeaB-Kp5.2, (b) StyD-1CP, and (c) StyD-CWB2 activities using different concentrations of phenylacetaldehyde. The assay was performed as mentioned in the “Materials and Methods” section. Various PA concentrations 0–1.0 (FeaB-Kp5.2), 0–1.5 (StyD-1CP), and 0–0.1 mmol L^-1 (StyD-CWB2) were applied, while the most suitable NAD⁺ concentration (FeaB-Kp5.2: 2.5; StyD-1CP: 6.0; StyD-CWB2: 2.0 mmol L^-1) was set corresponding to each enzyme. Data shown are averages of independently measured triplicates

Fig. 3

Relative residual activities of FeaB-Kp5.2 after incubation at temperatures between 0 and 50 °C in steps of 5 °C. The assay was performed as mentioned in the “Materials and Methods” section. The enzyme was heated up at the desired temperature and incubated for 30 min before it was investigated by the assay. Relative activities calculated by comparing of the residual activities to the initial activity (value at −20 °C; 8.4 0.3 U mg^-1) of the enzyme preparation are given. Data shown are averages of independently measured triplicates

Fig. 4

Phylogenetic analysis of various (phenylacetaldehyde) aldehyde dehydrogenases. The phylogenetic tree was calculated with MEGA6 using the minimum evolution method with 1000 bootstrap replications. Different StyDs originating from various pseudomonads as well as StyDs, FeaBs, and ALDHs from Rhodococcus opacus 1CP, Gordonia rubripertincta CWB2, Escherichia coli K-12 substr. DH10B, and Sphingopyxis fribergensis Kp5.2 are considered