Novel N-terminal amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), a mammalian monomeric enzyme. A partial N-terminal amino acid sequence of D-beta-hydroxybutyrate dehydrogenase from pig kidney was obtained by automated Edman degradation and confirmed by manual sequencing of the entire N-terminal portion of the holoenzyme, which is known to exist as a homodimer, and of cyanogen bromide fragments. This enzyme has a molecular weight of 62 000 Da as determined by gel filtration on Sephadex G-200 and a sedimentation coefficient of 4.5 S (20 degrees C). The sequence was as follows: NH2-MTR-GLI-SPP-LYS-HYI-MTR-ARG-LYS-ILE-LYS-LEU-ARG-HRS-THR-THR-SER-PRO-GLY-LYS-MET-ILE-GAP-PRG-THR-TYR-PHE-MET-ASP-GLY-VAL-ILE-GLY-LYS-ARG-LYS-GLY-ARG-ILE-LYS-THR-LEU-LEU-LEU-ILE-PRO-LEU-PRO-GLY-TYR-HIS-ARG-ASN-ASP-HIS-THR-LEU-HIS-LYS-LYS-ASN-SER-ASP-LEU-LEU-ASN-PRO-PRO-ASN-GLU-VAL-LYS-LYS-PRO-ASP-LYS-THR-PRO-ALA-PHE-SER-LEU-PHE-TYR-PHE-GLY-GLY-ASN-ILE-LEU-ASN-ILE-GLY. From these data, residues 1-56 represent the N-terminal amino acids of the mature protein, which was then characterized as a member of a new family of short-chain alcohol dehydrogenases. The N-terminal extension relative to the yeast L-lactate dehydrogenase (EC 1.1.1.28) includes amino acids found in all of the monomeric and dimeric D-beta-hydroxybutyrate dehydrogenases characterized to date (e.g. a leucine and a valine at the positions corresponding to 14 and 27, respectively). From the hydropathy plot of D-beta-hydroxybutyrate dehydrogenase, we suggest that residues 9-40 might represent a membrane-spanning domain and that residues 1-8 (part of the proposed signal peptide) or 15-35 could constitute a sequence of hydrophobic residues essential for enzyme assembly. The C-terminal portion of the D-beta-hydroxybutyrate dehydrogenase has about 25% homology with several enzymes from which the complete N-terminal sequence has been published, notably mammalian lactic acid dehydrogenase, D-isomer-specific D-beta-hydroxybutyrate dehydrogenase, mitochondrial L-lactate dehydrogenase and D-beta-hydroxybutyrate dehydrogenase from Drosophila melanogaster. Comparison with data on Drosophila D-beta-hydroxybutyrate dehydrogenase at the protein level reveals a close relationship between the enzymes from lower animals and higher eukaryotes, which supports the recently proposed phylogenetic relationship between the eucaryotic and invertebrate alpha-hydroxyacid dehydrogenases.