Characterization of a Bean (Phaseolus vulgaris L.) Malic‐enzyme Gene

Abstract

We have isolated a genomic clone encoding a plant NADP+-dependent malic enzyme (NADP-ME). This clone, isolated from bean (Phaseolus vulgaris L.), covers the entire gene (exons, introns) and 5'-flanking regions. DNA sequencing defines 20 exons spanning approximately 4.5 kb, which range over 48-235 bp in size. All 19 introns are fairly small (79-391 bp). The first intron resides in the S'hntranslated leader sequence. Introns 10, 11 and 16 are located at positions identical to a rat malic-enzyme gene. In the promoter region, a TATA box (TATATATA) is easily recognized 41 bp upstream of a single transcription-initiation site. Two potential cis-acting elements with homology to elements from plant genes, activated by UV light and fungal elicitors, were identified at positions -153 and -312, respectively. Southern-blot analysis suggests a single gene copy, but also other distantly related genes, in the bean genome. The deduced NADP-ME protein of 589 amino acids exhibits features consistent with a cytoplasmic location. We describe the organization of the NADP-ME protein into functional domains located on separate exons. The evolution of malicenzyme genes coding for isoforms in different cellular compartments of plants and animals is discussed.Malic enzyme [L-malate : NAD(P)-oxidoreductase (decarboxylating)] catalyzes the oxidative decarboxylation of malate to pymvate, generating reducing equivalents [NAD(P)H] and CO,. NAD+-dependent-malic-enzyme isoforms (NAD-ME) and isoforms using NADP' as a coenzyme (NADP-ME) are localized in different cellular compartments in both animals and plants [l, 21. Another form of NAD-ME, which, like NADP-ME, can also decarboxylate oxaloacetate is found predominantly in bacteria [3]. Malic enzyme is widely distributed and its activity is particularly high in specific metabolic situations. For example, it plays an important role in lipogenesis in mammalian liver, where a strictly cytosolic NADP-ME is linked to the generation of NADPH for fatty acid biosynthesis [l].Plants usually express nuclear genes for cytosolic NADPMEs and mitochondria1 NAD-MEs [2-51. A particular use of NADP-ME has evolved in C, plants such as maize, which employ a chloroplastic NADP-ME in a special mode of photosynthetic carbon fixation [6]. CO, is fixed into malate in mesophyll cells, shuttled into neighbouring bundle-sheath cells and released there from malate through chloroplastic Abbreviations. NAD-ME, NAD+ -dependent malic enzyme ; NADP-ME, NADP+-dependent malic enzyme; CAM, crassulaceanacid metabolism.Enzymes. NAD+ -dependent malic enzyme, oxaloacetate decarboxylating (EC 1.1 .I .38) ; NAD +-dependent malic enzyme (EC 1 .I .1.39) ; NADP+-dependent malic enzyme, oxaloacetate decarboxylating (EC 1.1.1.40).Note. The nucleotide sequences reported in this paper have been submitted to the GenbankEMBL Data Bank and are available under accession number X80051. tut fur Pflanzenphysiologie (260), D-70593 Stuttgart, Germany NADP-ME in order to be refixed by ribulose-1,s-bisphosphate carboxylase. Primary carbon fixation into m...

Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis

“…6). However, it actually encoded malic enzyme (24,25), being misidentified in part because of no biochemical confirmation. Other research groups used the malic enzyme cDNA in the mistaken belief that it encoded CAD, such as to study monolignol pathway induction in the rubber tree (Hevea brasiliensis; ref.…”

A pre-genetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides

“…The apparent universal possession of malic enzyme and the similarity, at the amino acid and DNA level, of malic enzymes from disparate sources (Hodnett et al, 1996 ;Viljoen et al, 1994 ;Walter et al, 1994) suggest a fundamental and crucial cellular function for this enzyme. Amongst the suggested functions for malic enzyme are roles in the metabolism of acetate (McCullough & Roberts, 1974), involvement in anaerobic growth (Boles et al, 1998), and generation of NADPH for lipid biosynthesis (Evans & Ratledge, 1985 ;Frenkel, 1975 ;Hodnett et al, 1996).…”

Primary Metabolism in Plant Defense (Regulation of a Bean Malic Enzyme Gene Promoter in Transgenic Tobacco by Developmental and Environmental Cues)

“…The deduced amino acid sequence of this cDNA, now redesignated cPvMEl, displays 73 to 79% sequence identity without gaps to other plant NADP-MEs, clearly demonstrating its true identity as a bean ME clone (Walter et al, 1994). The PvMEl protein does not contain an apparent signal peptide for translocation into chloroplasts, which strongly suggests that it represents a cytosolic isoform of NADP-ME.…”

“…The primary structure of severa1 plant NADP-MEs is now known from cDNA studies and has led to the identification of a NADP-ME cDNA clone from common bean (Phaseolus vulgaris L.), which was earlier erroneously believed to encode another NADP-dependent dehydrogenase, a cinnamyl-alcohol dehydrogenase (Walter et al, 1988(Walter et al, , 1990a(Walter et al, , 1994. The deduced amino acid sequence of this cDNA, now redesignated cPvMEl, displays 73 to 79% sequence identity without gaps to other plant NADP-MEs, clearly demonstrating its true identity as a bean ME clone (Walter et al, 1994).…”

“…This is in accordance with its origin from a C, species, which is considered to contain genes for cytosolic forms but not for chloroplastic NADP-MEs. Moreover, the N terminus contains none of the features described for mitochondrial targeting peptides (Von Heijne et al, 1989;Walter et al, 1994).…”