Structure and biochemistry of phenylacetaldehyde dehydrogenase from the Pseudomonas putida S12 styrene catabolic pathway.
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.
Department of Chemistry, Pomona College, Claremont, CA, United States.
Anders G
AG
Crabo
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, United States.
Baljit
B
Singh
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, United States.
Tim
T
Nguyen
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, United States.
Shahram
S
Emami
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, United States. Electronic address: gassner@sfsu.edu.
George T
GT
Gassner
Department of Chemistry, Pomona College, Claremont, CA, United States.
Matthew H
MH
Sazinsky
D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008968:Molecular Conformation; D009243:NAD / Q000737:chemistry; D000072417:Protein Domains; D011549:Pseudomonas; D016958:Pseudomonas putida / Q000201:enzymology*; D012756:Sheep; D013050:Spectrometry, Fluorescence; D020058:Styrene / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D000447:Aldehydes; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009243:NAD; D020058:Styrene; D000445:Aldehyde Oxidoreductases; C053855:phenylacetaldehyde dehydrogenase
Kinetics; Phenylacetaldehyde dehydrogenase; Structure; Styrene monooxygnease