New superfamily members identified for Schiff-base enzymes based on verification of catalytically essential residues.
Enzymes that utilize a Schiff-base intermediate formed with their substrates and that share the same alpha/beta barrel fold comprise a mechanistically diverse superfamily defined in the SCOPS database as the class I aldolase family. The family includes the "classical" aldolases fructose-1,6-(bis)phosphate (FBP) aldolase, transaldolase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. Moreover, the N-acetylneuraminate lyase family has been included in the class I aldolase family on the basis of similar Schiff-base chemistry and fold. Herein, we generate primary sequence identities based on structural alignment that support the homology and reveal additional mechanistic similarities beyond the common use of a lysine for Schiff-base formation. The structural and mechanistic correspondence comprises the use of a catalytic dyad, wherein a general acid/base residue (Glu, Tyr, or His) involved in Schiff-base chemistry is stationed on beta-strand 5 of the alpha/beta barrel. The role of the acid/base residue was probed by site-directed mutagenesis and steady-state and pre-steady-state kinetics on a representative member of this family, FBP aldolase. The kinetic results are consistent with the participation of this conserved residue or position in the protonation of the carbinolamine intermediate and dehydration of the Schiff base in FBP aldolase and, by analogy, the class I aldolase family.
Choi
Kyung H
KH
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118-2394, USA.
Lai
Vicky
V
Foster
Christine E
CE
Morris
Aaron J
AJ
Tolan
Dean R
DR
Allen
Karen N
KN
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D002384:Catalysis; D019143:Evolution, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012545:Schiff Bases / Q000737:chemistry; D016415:Sequence Alignment
D012545:Schiff Bases; D000446:Aldehyde-Lyases