Research Article: 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover

Date Published: February 3, 2017

Publisher: Public Library of Science

Author(s): Soraya Pornsuwan, Somchart Maenpuen, Philaiwarong Kamutira, Pratchaya Watthaisong, Kittisak Thotsaporn, Chanakan Tongsook, Maneerat Juttulapa, Sarayut Nijvipakul, Pimchai Chaiyen, Alessandro Giuffrè.

http://doi.org/10.1371/journal.pone.0171135

Abstract

3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k’cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

Partial Text

The biodegradation of aromatic compounds is essential for recycling carbon sources in nature. Lignin, one of the most abundant biomasses found in nature is composed of aromatic compounds in the forms of benzenoid and phenolic derivatives [1]. Enzymes involved in the process of lignin degradation are of interest in biorefinery for the conversion of low value biomass into valuable chemicals [2–8]. The biodegradation of phenolic acids is often initiated with ortho-hydroxylation to form catecholic compounds by flavin-dependent or metal-dependent hydroxylases [9–11]. The resulting catecholic derivatives are generally further converted into ring-cleaved products by dioxygenases that catalyze C-C bond breaking either adjacent to the vicinal OH substituents (‘extradiol’ cleavage) or between the two hydroxyl groups (‘intradiol’ cleavage) [12, 13]. The breakage of catecholic compounds is a key step in the conversion of stable aromatic derivatives into aliphatic acids that can be further assimilated by microbes. For biotechnology applications, pathways involving the oxidative degradation of aromatic compounds are valuable for the microbial production of useful chemicals such as succinic or lactic acid [7, 14].

3,4-Dihydroxyphenylacetate 2,3 dioxygenase from Pseudomonas aeruginosa (PaDHPAO) was overexpressed, purified and characterized for its enzymatic properties as summarized in Table 6. The results showed that PaDHPAO belongs to an enzyme cluster that is largely unexplored. Overexpression of the enzyme in E. coli BL21 (DE3) yielded ~221 mg per 3.2 liters of cell culture. Metal analysis and activity assays with enzyme reconstituted with various metals indicated that Fe(II) is the native cofactor of PaDHPAO and that the enzyme contains 1 Fe per subunit. The loss of PaDHPAO activity during purification could be restored by adding a mixture of Fe(NH4)2(SO4)2 and DTT. Ascorbic acid or ROS scavenging enzyme (catalase or superoxide dismutase) can be used as a preservative agent for maintaining the activity of activated PaDHPAO. Analysis of the enzyme product by 1H-NMR has clearly identified PaDHPAO as an extradiol ring cleavage 2,3-dioxygenase. PaDHPAO is specific to catecholic substrates with vicinal hydroxyl groups at the 3 and 4 positions. Furthermore, a single methylene moiety between the carboxylate and phenyl group is required for the most optimal activity. The optimum temperature and pH for PaDHPAO catalysis is 55°C and pH 7.5, respectively. As PaDHPAO and the other two enzymes in Cluster 1 all have high specific activities, it is possible that DHPAO members of this cluster are also dioxygenases with fast turnovers. As most of the enzymes in this cluster are largely unexplored, the results reported herein are useful for establishing an understanding of the catalytic properties of these dioxygenases.

 

Source:

http://doi.org/10.1371/journal.pone.0171135

 

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