Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

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First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2017.02.004 ◽

2017 ◽

Vol 124 (1) ◽

pp. 23-27 ◽

Cited By ~ 3

Author(s):

Ryushi Kawakami ◽

Haruhiko Sakuraba ◽

Taketo Ohmori ◽

Toshihisa Ohshima

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Pyrococcus Horikoshii ◽

Broad Substrate Specificity ◽

Amino Acid Racemase

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Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized l-N-carbamoylase and N-succinyl-amino acid racemase

Applied Microbiology and Biotechnology ◽

10.1007/s00253-014-5880-7 ◽

2014 ◽

Vol 99 (1) ◽

pp. 283-291 ◽

Cited By ~ 13

Author(s):

Pablo Soriano-Maldonado ◽

Francisco Javier Las Heras-Vazquez ◽

Josefa María Clemente-Jimenez ◽

Felipe Rodriguez-Vico ◽

Sergio Martínez-Rodríguez

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Kinetic Resolution ◽

Dynamic Kinetic Resolution ◽

Amino Acid Racemase

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Assaying d-Body Amino Acids as d-Alanine and Amino Acid Racemase (AARase) Activity Using NADH Oxidoreduction Enzymic System

Lactic Acid Bacteria - Methods in Molecular Biology ◽

10.1007/978-1-4939-8907-2_3 ◽

2018 ◽

pp. 23-31

Author(s):

Natsuki Matsumoto ◽

Makoto Kanauchi

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Racemase

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Cystathionine β-lyase is involved in d-amino acid metabolism

Biochemical Journal ◽

10.1042/bcj20180039 ◽

2018 ◽

Vol 475 (8) ◽

pp. 1397-1410 ◽

Cited By ~ 5

Author(s):

Tetsuya Miyamoto ◽

Masumi Katane ◽

Yasuaki Saitoh ◽

Masae Sekine ◽

Hiroshi Homma

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Acid Metabolism ◽

Environmental Changes ◽

Biosynthetic Pathways ◽

E Coli ◽

Comparable Level ◽

Multifunctional Enzymes ◽

Dehydratase Activity ◽

Amino Acid Racemase

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

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TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Journal of Bacteriology ◽

10.1128/jb.00617-20 ◽

2021 ◽

Author(s):

Ren-Chao Zheng ◽

Xia-Feng Lu ◽

Hiroya Tomita ◽

Shin-ichi Hachisuka ◽

Yu-Guo Zheng ◽

...

Keyword(s):

Amino Acid ◽

Surface Glycoprotein ◽

Lag Phase ◽

Class Iii ◽

Cell Surface Glycoprotein ◽

Hyperthermophilic Archaeon ◽

Thermococcus Kodakarensis ◽

Domains Of Life ◽

Amino Acid Racemase

Members of Thermococcales harbor a number of genes encoding putative aminotransferase Class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell-surface glycoprotein gene TK0895 (Pcsg). The purified protein did not display aminotransferase activity, but exhibited racemase activity. An examination on most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal media supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase, but displayed cell yield similar to that observed in medium with l-Leu. By contrast, the ΔTK1211 strain displayed growth in medium with l-Leu, but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells, and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. IMPORTANCE Phylogenetic analysis of aminotransferase Class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups can now be presumed to function as a broad substrate specificity amino acid racemase (Subgroup 1), alanine/serine racemase (Subgroup 2), ornithine ω-aminotransferase (Subgroup 3), or Leu/Met racemase (Subgroup 4).

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The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽

10.1128/mbio.01213-19 ◽

2019 ◽

Vol 10 (5) ◽

Author(s):

Yasuyuki Yamamoto ◽

Tamotsu Kanai ◽

Tsuyoshi Kaneseki ◽

Haruyuki Atomi

Keyword(s):

Amino Acids ◽

Transcriptional Regulation ◽

Amino Acid ◽

Aromatic Amino Acid ◽

Amino Acid Biosynthesis ◽

Acid Biosynthesis ◽

Content Type ◽

Hyperthermophilic Archaeon ◽

Thermococcus Kodakarensis ◽

Aromatic Amino Acid Biosynthesis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

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Amidohydrolase Process: Expanding the use of l-N-carbamoylase/N-succinyl-amino acid racemase tandem for the production of different optically pure l-amino acids

Process Biochemistry ◽

10.1016/j.procbio.2014.04.013 ◽

2014 ◽

Vol 49 (8) ◽

pp. 1281-1287 ◽

Cited By ~ 10

Author(s):

Pablo Soriano-Maldonado ◽

María José Rodríguez-Alonso ◽

Carmen Hernández-Cervantes ◽

Ignacio Rodríguez-García ◽

Josefa María Clemente-Jiménez ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Optically Pure ◽

Amino Acid Racemase

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Genome Sequence of Lactobacillus sakei LK-145 Isolated from a Japanese Sake Cellar as a High Producer of d-Amino Acids

Genome Announcements ◽

10.1128/genomea.00656-17 ◽

2017 ◽

Vol 5 (33) ◽

Cited By ~ 3

Author(s):

Shiro Kato ◽

Tadao Oikawa

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Lactobacillus Sakei ◽

Content Type ◽

Genes Encoding ◽

Amino Acid Racemase

ABSTRACT This announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of d-amino acids. Three putative genes encoding an amino acid racemase were identified.

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Dietary intake of tryptophan tied emotion-related impulsivity in humans

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000608 ◽

2019 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Florian Javelle ◽

Descartes Li ◽

Philipp Zimmer ◽

Sheri L. Johnson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Food Intake ◽

Essential Amino Acid ◽

Food Habits ◽

Psychological Disorder ◽

Serotonin Synthesis ◽

Amino Acid Tryptophan ◽

Pass Through ◽

Three Factor

Abstract. Emotion-related impulsivity, defined as the tendency to say or do things that one later regret during periods of heightened emotion, has been tied to a broad range of psychopathologies. Previous work has suggested that emotion-related impulsivity is tied to an impaired function of the serotonergic system. Central serotonin synthesis relies on the intake of the essential amino acid, tryptophan and its ability to pass through the blood brain barrier. Objective: The aim of this study was to determine the association between emotion-related impulsivity and tryptophan intake. Methods: Undergraduate participants (N = 25, 16 women, 9 men) completed a self-rated measure of impulsivity (Three Factor Impulsivity Index, TFI) and daily logs of their food intake and exercise. These data were coded using the software NutriNote to evaluate intakes of tryptophan, large neutral amino acids, vitamins B6/B12, and exercise. Results: Correlational analyses indicated that higher tryptophan intake was associated with significantly lower scores on two out of three subscales of the TFI, Pervasive Influence of Feelings scores r = –.502, p < . 010, and (lack-of) Follow-Through scores, r = –.407, p < . 050. Conclusion: Findings provide further evidence that emotion-related impulsivity is correlated to serotonergic indices, even when considering only food habits. It also suggests the need for more research on whether tryptophan supplements might be beneficial for impulsive persons suffering from a psychological disorder.

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