Molecular characterization of lysine 6-dehydrogenase from Achromobacter denitrificans

Ruldeekulthamrong, Prakarn; Maeda, Sayaka; Kato, Shin‐ichiro; Nagata, Shinji; Sittipraneed, Siriporn; Packdibamrung, Kanoktip; Misono, Haruo

doi:10.5483/bmbrep.2008.41.11.790

Cited by 2 publications

(3 citation statements)

References 18 publications

(29 reference statements)

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“…Especially, Lys 6-DH, it gave very strong protein band. The result was similar to the report of Ruldeekulthamrong in 2007 [57]. lysdh could overexpress even if it was not induced.…”

Section: Expression Of the Involving Genessupporting

confidence: 90%

“…So, our laboratory brought these pieces of knowledge to create E. coli strain, which could produce L-PA. This E. coli strain harbors lysdh encoding lysine-6dehydrogenase (Lys 6-DH, ADK) from Acromobacter denitrificans K-1 [24] and proC encoding pyrroline-5-carboxylate reductase (P5CR) [25]. The reactions which converting L-lysine into L-PA by the activities of Lys 6-DH and P5CR are shown in Figure 3.…”

Section: Figurementioning

confidence: 99%

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L-pipecolic acid production in thrA knockout Escherichia coli

Khuanwilai

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L-pipecolic acid (L-PA) is a non-proteinogenic amino acid, however, it is an important precursor and a key ingredient of many pharmaceutically compound syntheses such as immunosuppressants and anesthetics. The production of amino acids via fermentation of microorganisms are getting attention, one of the widely used is an engineered�E. coli. L-PA production in the engineered E. coli uses L-lysine in its cell as a substrate. This research aimed to increase L-PA production by thrA knockout. The thrA encodes homoserine dehydrogenase I, the enzyme which draws the intermediate in L-lysine biosynthesis pathway to synthesize L-threonine. The group II intron insertion was used for disruption of thrA by specific insertion at the target gene. The capability of group II intron insertion for thrA in E. coli (E. coli BL21(DE3) ?thrA) was 65%. Moreover, pE22-LPC*D* was constructed and transformed into�E. coli BL21(DE3) (namely W-LPCD) and E. coli BL21(DE3) ?thrA (namely KO-LPCD). pE22-LPC*D* consisted of lysdh (encoding for lysine 6-dehydrogenase) from Acromobacter denitrificans, proC (encoding for pyrroline-5-carboxylate reductase) from Bacillus cereus ATCC 11778, and homologous lysC* and� dapA* which encode for lysine feedback resistant aspartokinase and dihydrodipicolinate synthase, respectively. The highest L-PA production by KO-LPCD was 0.57 g/L when it was cultured in Ying medium containing glycerol as a carbon source at 198 hours after induction with 0.1 mM IPTG.�The specific L-PA production was 0.049 g/g WCW, which was 1.8-fold of that obtained from W-LPCD.

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“…Especially, Lys 6-DH, it gave very strong protein band. The result was similar to the report of Ruldeekulthamrong in 2007 [57]. lysdh could overexpress even if it was not induced.…”

Section: Expression Of the Involving Genessupporting

confidence: 90%

Section: Figurementioning

confidence: 99%

L-pipecolic acid production in thrA knockout Escherichia coli

Khuanwilai

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“…catalyzes the oxidative deamination of the ⑀-amino group of L-lysine to form L-2-aminoadipate 6-semialdehyde, which in turn nonenzymatically cyclizes to form ⌬ 1 -piperideine-6-carboxylate (P6C) (1)(2)(3). The second is L-lysine 2-dehydrogenase (EC 1.4.1.15), which catalyzes the oxidative deamination of the ␣-amino group of L-lysine in the same way other amino acid dehydrogenases do (4).…”

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confidence: 99%

First Crystal Structure of l-Lysine 6-Dehydrogenase as an NAD-dependent Amine Dehydrogenase

Yoneda

Junya

Sakuraba

et al. 2010

Journal of Biological Chemistry

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A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 min at 100°C. The product of the enzyme's catalytic activity is ⌬ 1 -piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ⑀-amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 Å in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.L-Lysine dehydrogenase catalyzes the oxidative deamination of L-lysine in the presence of NAD. To date, two types of L-lysine dehydrogenase have been identified (Fig. 1). The first is L-lysine 6-dehydrogenase (LysDH 2 ; EC 1.4.1.18). This enzyme catalyzes the oxidative deamination of the ⑀-amino group of L-lysine to form L-2-aminoadipate 6-semialdehyde, which in turn nonenzymatically cyclizes to form ⌬ 1 -piperideine-6-carboxylate (P6C) (1-3). The second is L-lysine 2-dehydrogenase (EC 1.4.1.15), which catalyzes the oxidative deamination of the ␣-amino group of L-lysine in the same way other amino acid dehydrogenases do (4). With respect to the latter, there has been one report of an L-lysine 2-dehydrogenase catalyzing the deamination of the ␣-amino group in humans (5), but because the reaction product of this liver enzyme has not yet been identified, it is still unclear whether or not the enzyme actually catalyzes the oxidative deamination of L-lysine at the ␣-amino group. By contrast, LysDH has been identified in several microorganisms, and some of those enzymes have been characterized. The first known LysDH was found in Agrobacterium tumefaciens, where it plays a key role in L-lysine metabolism (1). Since then, this enzyme has been extensively characterized (6 -9) and utilized for assaying L-lysine (9).Recently, the gene encoding LysDH in Geobacillus stearother...

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Molecular characterization of lysine 6-dehydrogenase from Achromobacter denitrificans

Cited by 2 publications

References 18 publications

L-pipecolic acid production in thrA knockout Escherichia coli

L-pipecolic acid production in thrA knockout Escherichia coli

First Crystal Structure of l-Lysine 6-Dehydrogenase as an NAD-dependent Amine Dehydrogenase

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