Characterization of Plastid 5-Aminolevulinate Dehydratase (ALAD; EC 4.2.1.24) from Spinach (Spinacia olevacea L.) by Sequencing and Comparison with Non-Plant ALAD Enzymes

Abstract

We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant. A fulllength 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 Mg2+-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.

Purification, Metal Cofactor, N‐Terminal Sequence and Subunit Composition of a 5‐Aminolevulinic Acid Dehydratase from the Unicellular Green Alga Scenedesmus obliquus, Mutant C‐2A′

“…The consensus sequence reads DXXXDXXXXDGHDXX. This sequence was found for the Alv-D from Chlamydomonas reinhardtii (Matters and Beale, 1995), spinach (Schaumburg et al, 1992), pea (Boese et al, 1991), soybean (Kaczor et al, 1994), S. martensii (Schaumburg et al, 1992) and tomato (Polking et al, 1995) In 1968 an Ah-D from Rhodopseudonzonas spheroides was described which is K+-dependent. Recently, a possible third group of A h -D was found showing some features of both types of metal-binding domains.…”

“…These values are quite similar to those obtained for the enryme from spinach with 324 kDa for the native cnzyme and 45-50 kDa for the subunit (Liedgens et al, 1983). The seqw-nce-derived value for the spinach subunit was shown to be 40 123 Da (Schaumburg et al, 1992). The enzymes from various mammalian sources (human erythrocytes, Anderson and Desnick, 1979 ; beef liver, Bevan et al, 1980;Jordan and Seehra, 1986) and yeast (Borralho et al, 1990) show, in contrast, a lower molecular mass of about 37 kDa for the subunit at higher masses for the native enzymes ranging from 340 kDa to 350 kDa.…”

“…Furthermore, it is not senxitive to oxygen. The exact molecular mass of its subunits was calculated from the nucleotide sequence to be 40123 Da (Schaumburg et al, 1992). From a variety of other plant source"; the primary structure of A h -D was also determined, e.g.…”

Chlorophyll Biosynthesis.

“…lsotopically labeled substrates reveal that the initially bound 5-aminolevulinic acid forms a Schiff base with its keto group and the 8-amino group of a lysine at position 247 of the enzyme (E. coli numbering) that, along with an adjacent proline, is conserved in all 16 dehydratases whose sequence is known. These enzymes include representatives from mammals, bacteria, yeast, and plants, including spinach (Schaumburg et al, 1992), pea (Boese et al, 1991) porphobilinogen, whereas the second molecule of 5-aminolevulinate (which gives rise to the acetic acid side chain) is placed in a separate substrate binding site that allows the aldo1 condensation between the C3 of this second molecule and the C4 of the first molecule to proceed. Condensation is accompanied by detachment from the enzyme's lysine nitrogen.…”

A cDNA Clone for 5-Aminolevulinic Acid Dehydratase from Tomato (Lycopersicon esculentum Mill.)

“…cDNAs encoding ALAD have been cloned from pea (Boese et al, 1991), spinach (Schaumburg et al, 1992), and soybean (Kaczor et al, 1994) in addition to those isolated from animal and bacterial species. We report here the complete nucleotide sequence of a cDNA for tomato fruit ALAD ( Table I).…”