Structure and biochemistry of phenylacetaldehyde dehydrogenase from the Pseudomonas putida S12 styrene catabolic pathway

Abstract

Phenylacetaldehyde dehydrogenase catalyzes the NAD⁺-dependent oxidation of phenylactealdehyde to phenylacetic acid in the styrene catabolic and detoxification pathway of Pseudomonas putida (S12). Here we report the structure and mechanistic properties of the N-terminally histidine-tagged enzyme, NPADH. The 2.83 Å X-ray crystal structure is similar in fold to sheep liver cytosolic aldehyde dehydrogenase (ALDH1), but has unique set of intersubunit interactions and active site tunnel for substrate entrance. In solution, NPADH occurs as 227 kDa homotetramer. It follows a sequential reaction mechanism in which NAD⁺ serves as both the leading substrate and homotropic allosteric activator. In the absence of styrene monooxygenase reductase, which regenerates NAD⁺ from NADH in the first step of styrene catabolism, NPADH is inhibited by a ternary complex involving NADH, product, and phenylacetaldehyde, substrate. Each oligomerization domain of NPADH contains a six-residue insertion that extends this loop over the substrate entrance tunnel of a neighboring subunit, thereby obstructing the active site of the adjacent subunit. This feature could be an important factor in the homotropic activation and product inhibition mechanisms. Compared to ALDH1, the substrate channel of NPADH is narrower and lined with more aromatic residues, suggesting a means for enhancing substrate specificity.

Keywords: Kinetics; Phenylacetaldehyde dehydrogenase; Structure; Styrene monooxygnease.

Figure 1

The Styrene Catabolic Pathway of P. putida.

Figure 2

Global fold of PADH. A) Surface representation of the PADH dimer depicting the location of the substrate channel (*). B) PADH tetramer. Orientation and proximity of the oligomerization domain to the substrate channel for C) PADH (yellow) and BcPADH (blue) and D) ALDH1.

Figure 3

A) Stereoview of the PADH active site and substrate channel. NAD⁺ (green) was modeled in into the apo-PADH structure using the existing structure of ALDH1 with bound NAD⁺ (1BXS). Because E267 is disordered in PADH, E267 from BcPADH (yellow) is depicted in its place to provide a likely model of the apo-active site. B) Active site water molecule in BcPADH.

Figure 4

The Proposed Catalytic Mechanism of PADH.

Figure 5

Steady state reaction of NPADH in the presence or absence of product inhibitors. Reactions were carried out at 25°C in 50 mM POPSO buffer pH 8 containing 120 μM PAL and 0.4 μM enzyme. Reactions included either no inhibitor (❍), 95.2 mM phenylacetic acid (□), or 100 μM NADH (◇).

Figure 6

The binding of pyridine nucleotides to NPADH as monitored by fluorescence. Assays were performed in a 20 mM POPSO, pH 7.0 buffer with 2 mM Mg²⁺ at 25 °C. (A) Increase in fluorescence emission monitored at 469 nm in the titration of 8.8 μM NPADH with NADH. (B) Decrease in 469 nm fluorescence emission observed in the titration 7.0 μM NPADH equilibrated with 50 μM NADH with NAD⁺. Samples were excited at 340 nm and all fluorescence data were corrected for the inner filter effect. Equations 3a and 3b were used to generate fits through the data points in panels A and B, respectively.

Figure 7

Activating and inhibitory effects of divalent metal ions on steady-state catalysis by NPADH. A) The rate of NADH production by 0.1 μM NPADH reacting with 10 μM PAL and 50 μM NAD⁺ in 50 mM POPSO buffer at pH 7 in the presence (□) or absence (■) of 50 μM NADH as a function of (A) Mg²⁺ and (B) Mn²⁺. Best fits through the data according to equation 4 are represented by the solid curves passing through the plotted data.

Figure 8

Global fitting of the steady state NPADH reaction as a function NAD⁺ and PAL concentration. NAD⁺ concentrations (increasing from the lowest curve to the highest curve) were 50, 100, 150, 250, and 500 μM. Initial rates from the reaction of 1 μM NPADH with various concentrations of PAL and NAD⁺ in a 50 mM POPSO, 2 mM Mg²⁺ buffer at pH 7 were globally fit according to the model shown in (Scheme S4) by using equation 5b. The fit converged with a global R² value of 0.99 and absolute sum of squares of 59.5.

Figure 9

Effect of SMOB on NADH inhibition. Reactions were carried out with 1 μM NPADH, 1 mM NAD⁺ and increasing amounts of PAL in 50 mM POPSO, 2 mM Mg²⁺ buffer at pH 7 in the presence (●) or absence (❍) of 1 μM SMOB, 30 μM FAD, and 40 μM cytochrome c.

Scheme 1

Summary of Steady-State Reactions Catalyzed by NPADH