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Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum

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Abstract

The genes encoding putative L-aspartate dehydrogenases (EC 1.4.1.21, ADH) from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum were cloned and expressed in Escherichia coli. The respective enzymes in the form of hybrid proteins with N-terminal hexahistidine tags were purified to apparent homogeneity. Both enzymes catalyzed in vitro the reductive amination of oxaloacetate to L-aspartate by an order faster than the reverse reaction at a respective pH optimum of 8.0–9.0 and 9.8; also, the enzymes only catalyzed amination under physiological conditions (pH 7.0–8.0). Their specificity to NADPH was higher by 1–2 orders of magnitude than that to NADH. The apparent K M values of ADH from R. palustris for oxaloacetate, ammonium, and NADPH at pH 9.0 were 9.2, 11.3, and 0.21 mM, respectively, and the corresponding K M values of ADH from B. japonicum were 21, 4.3, and 0.032 mM, respectively. The amination activity of novel ADHs may be important for the fixation of inorganic nitrogen in vivo and used for the construction of a bacterial strain-producer of L-aspartate by metabolic engineering methods.

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Abbreviations

ADH:

L-aspartate dehydrogenase

L-Asp:

L-aspartic acid

L-Glu:

L-glutamic acid

OA:

oxaloacetate

AAT:

aspartate aminotransferase

GOGAT:

glutamate synthase

GS:

glutamine synthase

GDH:

glutamate dehydrogenase

ADHtma:

ADH from Thermotoga maritima

ADHafu:

ADH from Archaeoglobus fulgidus

ADHreu :

ADH from Ralstonia eutropha

ADHpae :

ADH from Pseudomonas aeruginosa

ADHrpa :

ADH from Rhodopseudomonas palustris

ADHbja :

ADH from Bradyrhizobium japonic um

His:

histidine

Arg:

arginine

Ile:

isoleucine

Pro:

proline

ORF:

open reading frame

Ap:

ampicillin

IPTG:

isopropyl-β-D-1-thiogalactopyranoside

α-kG:

α-ketoglutarate

PEP:

phosphoenolpyruvate

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Authors and Affiliations

  1. Ajinomoto-Genetika Research Institute, Pervyi Dorozhnyi proezd 1-1, Moscow, 117545, Russia

    T. M. Kuvaeva, J. I. Katashkina, A. D. Kivero & S. V. Smirnov

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  1. T. M. Kuvaeva
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  2. J. I. Katashkina
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  3. A. D. Kivero
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  4. S. V. Smirnov
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Correspondence to T. M. Kuvaeva.

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Original Russian Text © T.M. Kuvaeva, J.I. Katashkina, A.D. Kivero, S.V. Smirnov, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 2, pp. 155–164.

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Kuvaeva, T.M., Katashkina, J.I., Kivero, A.D. et al. Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum . Appl Biochem Microbiol 49, 136–143 (2013). https://doi.org/10.1134/S0003683813010109

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