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-termininally histidine-tagged enzyme, NPADH. The 2.83Å 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.

A Review: The Styrene Metabolizing Cascade of Side-Chain Oxygenation as Biotechnological Basis to Gain Various Valuable Compounds

“…This is also the reason for a widespread occurrence of PAD genes in many organisms, but only a few studies are available about this enzyme class ( Tischler, 2015 ). Until now, only a few microbial PADs have been investigated including these of P. fluorescens ST ( Beltrametti et al, 1997 ), Pseudomonas putida S12 ( Crabo et al, 2017 ), E. coli K-12 ( Parrott et al, 1987 ; Ferrández et al, 1997 ; Hanlon et al, 1997 ; Zimmerling et al, 2017 ), Arthrobacter globisformis ( Shimizu et al, 1993 ), Xanthobacter sp. 124X ( Hartmans et al, 1989 ), Rhodococcus rhodochrous ( Hartmans et al, 1990 ) and the yeast-like fungus Exophiala jeanselmei ( Cox et al, 1996 ).…”

“… Beltrametti et al (1997) have revealed a strong homology between the StyD of P. fluorescens ST and other prokaryotic as well as even eukaryotic aldehyde dehydrogenases (ALDH, EC 1.2.1). The recently studied PAD of P. putida S12 was characterized as an N -terminal his-tagged protein and showed a structural similarity to sheep liver cytosolic aldehyde dehydrogenase (ALDH1) ( Crabo et al, 2017 ). These facts confirm the ubiquitous appearance of PADs due to their important role in metabolisms ( Liu et al, 1997 ).…”

“…Their active site containing of cysteine and glutamate is highly conserved and hence, the following catalytic mechanism is similar in all ALDHs ( Hempel et al, 1993 ; Liu et al, 1997 ). The catalytic relevant cysteine binds temporarily on the carbon atom of aldehyde’s carbonyl group to release the hydride ion of this group which is transferred to NAD(P) + ( Liu et al, 1997 ; Crabo et al, 2017 ). Then, the essential water molecule is attacked by negatively charged glutamate and the remaining hydroxide ion binds on the carbon atom of the carbonyl group.…”

“…In the last step, cysteine’s sulfur ion is unattached and deprotonated by glutamate’s acid group to form a thiol group. Finally, the aldehyde is converted into an acid, both amino acids are unaltered, one water molecule is consumed and the co-substrate is reduced to NAD(P)H ( Crabo et al, 2017 ).…”

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

“…A styrene monooxygenase (SMO) produces (S)-styrene oxide, which is converted by a membrane-bound styrene oxide isomerase (SOI) to phenylacetaldehyde. A phenylacetaldehyde dehydrogenase (PAD) oxidizes the aldehyde to phenylacetic acid (PAA) (7). PAA is a central catabolite and metabolized in the so-called lower degradation pathway, which is present in about 16% of all genome-sequenced microorganisms (8,9).…”