Characterization of plastid 5-aminolevulinate dehydratase (ALAD; EC 4.2.1.24) from spinach (Spinacia oleracea L.) by sequencing and comparison with non-plant ALAD enzymes.
We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant. A full-length cDNA clone (1727 bp) encoding this enzyme has been identified by immunoscreening a lambda gt 11 cDNA library of spinach. ALAD is not a plant-specific enzyme; however, the plant enzyme differs from the well known ALAD enzymes of bacteria, yeast and animals in structural and biochemical properties and in that it is located in the plastid. Differences and homologies can be traced back to the molecular level. The mature ALAD subunit, whose N-terminus was determined by automatic Edman degradation, is a protein of 367 amino acid residues and has a Mr of 40,132. This figure is in the range of molecular weights of non-plant ALADs. The active centre is highly conserved and the same is true for the ion-binding domain, except that 4 cysteines of the non-plant enzymes (binding Zn2+) have disappeared and a total of 6 aspartic acids meets the demands of Mg(2+)-binding. However, there are more distinct differences. Apart from a transit sequence of 56 amino acids targeting the plastid, the N-terminal part of the mature plant enzyme differs considerably from non-plant ALAD enzymes. It is rich in prolines and hydroxylated amino acids. The apparent Mr on SDS-PAGE is 45,000 or higher, but up to now posttranslational modifications have not been found.
Schaumburg
A
A
Botanisches Institut, Universität zu Köln.
Schneider-Poetsch
H A
HA
Eckerskorn
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D000623:Porphobilinogen Synthase / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011061:Poly A; D000623:Porphobilinogen Synthase