Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily.
Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs.
Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454,USA.
Megan P D
MPD
Zahniser
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), 402 N. Blackford Street, Indianapolis, IN 46202,USA.
Shreenath
S
Prasad
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), 402 N. Blackford Street, Indianapolis, IN 46202,USA.
Malea M
MM
Kneen
Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA.|Rosenstiel Basic Medical Sciences Research Center, MS029, 415 South Street, Waltham, MA 02454, USA.
Cheryl A
CA
Kreinbring
Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA.|Rosenstiel Basic Medical Sciences Research Center, MS029, 415 South Street, Waltham, MA 02454, USA.
Gregory A
GA
Petsko
Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA.|Rosenstiel Basic Medical Sciences Research Center, MS029, 415 South Street, Waltham, MA 02454, USA.|Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA.
Dagmar
D
Ringe
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), 402 N. Blackford Street, Indianapolis, IN 46202,USA.
Michael J
MJ
McLeish
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D051381:Rats
D009249:NADP; D000445:Aldehyde Oxidoreductases; C027015:benzaldehyde dehydrogenase (NAD+)
NAD(P)+; X-ray; hydride transfer; isotope effect; mutagenesis