Malate dehydrogenase from the mesophile Chlorobium vibrioforme and from the mild thermophile Chlorobium tepidum: molecular cloning, construction of a hybrid, and expression in Escherichia coli

Abstract

The genes (mdh) encoding malate dehydrogenase (MDH) from the mesophile Chlorobium vibrioforme and the moderate thermophile C. tepidum were cloned and sequenced, and the complete amino acid sequences were deduced. When the region upstream of mdh was analyzed, a sequence with high homology to an operon encoding ribosomal proteins from Escherichia coli was found. Each mdh gene consists of a 930-bp open reading frame and encodes 310 amino acid residues, corresponding to a subunit weight of 33,200 Da for the dimeric enzyme. The amino acid sequence identity of the two MDHs is 86%. Homology searches using the primary structures of the two MDHs revealed significant sequence similarity to lactate dehydrogenases. A hybrid mdh was constructed from the 3 part of mdh from C. tepidum and the 5 part of mdh from C. vibrioforme. The thermostabilities of the hybrid enzyme and of MDH from C. vibrioforme and C. tepidum were compared.Malate dehydrogenase (MDH) catalyzes the reversible conversion of malate to oxaloacetate, using NAD as cofactor. In most organisms, MDH is an essential enzyme in carbon metabolism, and in green sulfur bacteria, it is part of the reductive tricarboxylic acid cycle (9).Complete amino acid sequence data for MDH are available from many species within the Bacteria, Archaea, and Eucarya. Based on sequence similarity, Goward and Nicholls (15) have suggested that MDH has diverged into two distinct phylogenetic groups, one group that includes cytoplasmic MDH, chloroplast MDH, and MDH from Thermus flavus, and a second group that includes MDHs with similarity to lactate dehydrogenase (LDH). In the latter group, the mitochondrial MDH and eubacterial MDH are in one cluster and the more LDHlike (archaeal) Haloarcula marismortui MDH is in another.Although the primary structures of the various MDHs and LDHs are different, their tertiary structures are similar. The dehydrogenases have two domains; the structurally conserved N-terminal dinucleotide-binding domain, and the C-terminal catalytic domain, which differs among the different dehydrogenases. The dinucleotide-binding domain of the LDHs and MDHs that use NAD as a cofactor contains a characteristic glycine motif. In LDH, this is GXGXXG, whereas for most known MDHs, it is GAXGXX(G/A). However, MDHs from all photosynthetic bacteria studied so far have the glycine motif typical of LDH (4, 29).The amino acid residues important for substrate and coenzyme binding, catalysis, and subunit assembly have been identified in several MDHs and LDHs (3,27,36,42). With regard to thermostability, LDH from Bacillus species has been extensively studied (21,41,45,46), and studies have also been performed with MDH from Escherichia coli (14) and from the thermophilic Thermus aquaticus (6).In the present work, MDH and its corresponding gene from the two green sulfur bacteria Chlorobium tepidum and Chlorobium vibrioforme have been isolated, characterized, and compared. The green sulfur bacteria comprise a distinct phylogenetic group of photosynthetic species, which are related to other gro...

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

“…One group includes cytoplasmic MDH, chloroplast MDH, and MDH from Thermus flavus; the other group includes MDHs that are similar to lactate dehydrogenase (LDH) (Naterstad et al, 1996). This review is a comprehensive overview of the properties, function, crystallization, and evolution of the microbial MDHs.…”

Regulation of the mdh-sucCDAB operon in Rhizobium leguminosarum

“…A putative ribosome binding site (AGGA) is present 10 bp upstream of the translational start. Conserved residues known to bind substrate and participate in catalysis are found at Arg-83, Arg-89, Asp-149, Arg-152 His-176 and Ala-231 [11]. Residues 10^15 contain the glycine motif GXGXXG known to be characteristic of lactate dehydrogenases and the lactate-like Mdhs [12].…”

The putative l‐lactate dehydrogenase from Methanococcus jannaschii is an NADPH‐dependent l‐malate dehydrogenase

“…Crystallographic studies have shown that a charge imbalance inside the catalytic vacuole is responsible for substrate discrimination (Chapman et al ., 1999). Various data, based on the primary structure determination (Cendrin et al ., 1993; Naterstad et al ., 1996; Synstad et al ., 1996; Langelandsvik et al ., 1997), solution studies (Bonneté et al ., 1993), folding and association pathways (Madern et al ., 2000) and crystallographic studies (Richard et al ., 2000), have shown that, within the l ‐MalDH family, a lactate dehydrogenase‐like (LDH‐like) group of enzymes should be considered.…”