Publications

• Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex.

  Yoneda K., Sakuraba H., Tsuge H., Katunuma N., Ohshima T.

  The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resoluti ... >> More

  The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH. << Less

  FEBS J. 274:4315-4325(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Marine Proteobacteria metabolize glycolate via the beta-hydroxyaspartate cycle.

  Schada von Borzyskowski L., Severi F., Krueger K., Hermann L., Gilardet A., Sippel F., Pommerenke B., Claus P., Cortina N.S., Glatter T., Zauner S., Zarzycki J., Fuchs B.M., Bremer E., Maier U.G., Amann R.I., Erb T.J.

  One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments¹. Although it is generally accepted that glycolate is oxidized to glyoxyla ... >> More

  One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments¹. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria^2-4, the further fate of this C₂ metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the β-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago⁵. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing-to our knowledge-the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton. << Less

  Nature 575:500-504(2019) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• The first archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization.

  Yoneda K., Kawakami R., Tagashira Y., Sakuraba H., Goda S., Ohshima T.

  A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about ... >> More

  A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species. << Less

  Biochim. Biophys. Acta 1764:1087-1093(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

  Li Y., Kawakami N., Ogola H.J., Ashida H., Ishikawa T., Shibata H., Sawa Y.

  L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, c ... >> More

  L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production. << Less

  Appl Microbiol Biotechnol 90:1953-1962(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Aspartate dehydrogenase, a novel enzyme identified from structural and functional studies of TM1643.

  Yang Z., Savchenko A., Yakunin A.F., Zhang R., Edwards A.M., Arrowsmith C.H., Tong L.

  The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position ... >> More

  The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6A resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD(+) cofactor and a C-terminal alpha+beta domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases. << Less

  J. Biol. Chem. 278:8804-8808(2003) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• L-aspartate dehydrogenase: features and applications.

  Li Y., Ogola H.J., Sawa Y.

  L-amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of L-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors. These enzymes hav ... >> More

  L-amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of L-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member L-aspartate dehydrogenase (L-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of L-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type L-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few L-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes. << Less

  Appl Microbiol Biotechnol 93:503-516(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.