Cloning, structural analysis and expression of the gene encoding aspartate aminotransferase from the thermophilic cyanobacterium Phormidium lapideum

Kim, Hyeung; Nakaoka, Masaki; Yagi, Mayumi; Ashida, Hiroyuki; Hamada, Kensaku; Shibata, Hitoshi; Sawa, Yousuke

doi:10.1016/s1389-1723(03)80081-6

Cited by 5 publications

(6 citation statements)

References 12 publications

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“…Okamoto et al [6] reported that the high Pro content of the Ib-type AAT from T. thermophilus (6.5%) will render the enzyme rigid and thermostable. The same features are also found in other subgroup Ib AATs, such as Thermus aquaticus YT1 AAT (7.0%) [31] and Phormidium lapdideum (6.1%), as well as the newly found Ib-prokaryote-type AAT in Pinus pinaster (6.4%) [9,11]. The Pro content of B. subtilis B3 AAT is 4.1%, which is similar to that of subgroup Ia E. coli AAT (3.8%) and is much lower than that of subgroup Ib T. thermophilus AAT.…”

Section: Discussionsupporting

confidence: 70%

“…Up until now, the most extensively investigated AATs, with studies reported on their structure as well as their function, are those from subgroup Ia, whereas much less is known about AATs from subgroup Ib. Recently, the 3D structures of the subgroup Ib AATs from T. thermophilus, Phormidium lapideum and Thermotoga maritima were solved, showing that the structures of the enzymes in subgroups Ia and Ib are very similar [10][11][12] and that the active site residues are well-conserved [6].…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

Wu¹,

Yang²,

Wang³

et al. 2011

The FEBS Journal

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In the present study, we report the identification of a new gene from the Bacillus subtilis B3 strain (aatB3), which comprises 1308 bp encoding a 436 amino acid protein with a monomer molecular weight of 49.1 kDa. Phylogenetic analyses suggested that this enzyme is a member of the Ib subgroup of aspartate aminotransferases (AATs; EC 2.6.1.1), although it also has conserved active residues and thermostability characteristic of Ia‐type AATs. The Asp232, Lys270 and Arg403 residues of AATB3 play a key role in transamination. The enzyme showed maximal activity at pH 8.0 and 45 °C, had relatively high activity over an alkaline pH range (pH 7.0–9.0) and was stable up to 50 °C. AATB3 catalyzed the transamination of five amino acids, with l‐aspartate being the optimal substrate. The Km values were determined to be 6.7 mm for l‐aspartate, 0.3 mm for α‐ketoglutarate, 8.0 mm for l‐glutamate and 0.6 mm for oxaloacetate. A 32‐residue N‐terminal amino acid sequence of this enzyme has 53% identity with that of Bacillus circulans AAT, although it is absent in all other AATs from different organisms. Further studies on AATB3 may confirm that it is potentially beneficial in basic research as well as various industrial applications. Database  The nucleotide sequence data have been deposited in the GenBank database under accession Numbers

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Section: Discussionsupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

Wu¹,

Yang²,

Wang³

et al. 2011

The FEBS Journal

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“…Few studies, using mainly site-directed mutagenesis, have shown that the character of the residues in AAT polypeptide chain adjacent to those binding cofactor and substrate also influence the enzyme catalytic efficiency [14][15][16]. It seems that substitutions within these residues might be crucial for the differences in the kinetic parameters values (k cat or k cat /K m ) determined for recombinant AAT of several origins: bacterial [17], animal [18], plant [19] and native enzymes from three different bacteria species [16,20,21], or from sheep's liver [22].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

2013

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Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

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“…Here, we found that the Pro content of NhAAT is 6.1%, which may be the reason why the NhAAT has a higher optimum temperature of 45℃. The optimum temperature of AAT from P. lapdideum is 35 °C higher than the optimum growth temperature of P. lapdideum [ 22 ]. This shows that the optimal reaction temperature of AAT is not necessarily close to the optimal growth temperature of organisms.…”

Section: Discussionmentioning

confidence: 99%

“…The calculated K m values of NhAAT was 5.67 and 6.16 mM for L-glutamic acid and L-aspartic acid, respectively. The K m values of NhAAT for L-glutamic acid was close to that of cyanobacteria Phormidium lapideum (5.7 mM) [ 22 ], and is lower than that of alfalfa Medcago sativa L. (18.5 mM) [ 27 ]. The K m values of NhAAT for L-aspartic acid was close to that of Bacillus subtilis B3 (6.68 mM) [ 10 ] and Trypanosoma brucei (6.8 mM) [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

Isolation and functional verification of an aspartate aminotransferase gene from Neoporphyra haitanensis

Shao

et al. 2023

BMC Plant Biol

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Background Neoporphyra haitanensis is a commercial laver species in China. Aspartic acid is an important flavor amino acid, and aspartate aminotransferase (AAT) is a crucial enzyme in its biosynthesis. In this study, we cloned one AAT gene (NhAAT) from the red alga N. haitanensis and investigated its sequence structure, transcriptional expression and enzymatic characteristics. The purpose of our research is to obtain a functional AAT responsible for the biosynthesis of aspartic acid from red seaweeds, which has the potential to influence the flavor of N. haitanensis. Results Sequence analysis showed that NhAAT contains a conserved domain of Aminotran_1_2, which belongs to the transaminase superfamily. The secondary structure of NhAAT is dominated by α-helix. The results of enzymatic characterization illustrated that the NhAAT has highest catalytic activity at 45 °C and pH 7.5 in both forward and reverse reactions. The calculated Km values of NhAAT was 5.67 and 6.16 mM for L-glutamic acid and L-aspartic acid, respectively. Quantitative analysis showed that the NhAAT expression of N. haitanensis collected in late harvest (Dec) was 4.5 times that of N. haitanensis collected in early harvest (Oct), while the aspartic acid content of N. haitanensis collected in late harvest (Dec) was 1.2 times that of N. haitanensis collected in early harvest (Oct). Conclusion The results of enzyme kinetics indicated that NhAAT prefers to catalyze the reaction in the direction of aspartic acid production. Moreover, the trend of NhAAT expression level was consistent with that of aspartic acid content in N. haitanensis in different harvest periods. Our research is helpful to understand the accumulation and regulation of amino acids in N. haitanensis in different habitats and the taste difference of N. haitanensis in different harvest periods.

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Cloning, structural analysis and expression of the gene encoding aspartate aminotransferase from the thermophilic cyanobacterium Phormidium lapideum

Cited by 5 publications

References 12 publications

Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Isolation and functional verification of an aspartate aminotransferase gene from Neoporphyra haitanensis

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