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Crystal structure and active site location of N-(1-D-carboxylethyl)-L-norvaline dehydrogenase

Nature Structural Biology volume 5, pages 593–601 (1998)Cite this article

Abstract

Opine dehydrogenases catalyze the NAD(P)H-dependent reversible reaction to form opines that contain two asymmetric centers exhibiting either (L,L) or (D,L) stereochemistry. The first structure of a (D,L) superfamily member, N-(1-D-carboxylethyl)-L-norvaline dehydrogenase (CENDH) from Arthrobacter sp. strain 1C, has been determined at 1.8 Å resolution and the location of the bound nucleotide coenzyme has been identified. Six conserved residues cluster in the cleft between the enzyme's two domains, close to the nucleotide binding site, and are presumed to define the enzyme's catalytic machinery. Conservation of a His-Asp pair as part of this cluster suggests that the enzyme mechanism is related to the 2-hydroxy acid dehydrogenases. The pattern of sequence conservation and substitution between members of this enzyme family has permitted the tentative location of the residues that define their differential substrate specificities.

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Figure 1: Stereo diagrams, prepared using MOLSCRIPT49, of a single subunit of CENDH.
Figure 2: Stereo representation of a CENDH dimer viewed with the two-fold axis horizontal and prepared using MOLSCRIPT.
Figure 3: The intersubunit interface of the CENDH dimer, viewed across the two-fold axis drawn using the program MIDASPLUS50.
Figure 4: a, A BOBSCRIPT51 representation of the positive difference electron density calculated with coefficients (|FND1| - |FNA1|)αiso, contoured at 1σ for the binary complex of CENDH with NAD+.
Figure 5: Structure-based alignment of the sequence for the CENDH from Arthrobacter sp. strain 1C together with the other two sequenced (D,L) opine dehydrogenases: nopaline dehydrogenase (DHNO) and lysopine dehydrogenase (DHLO) both from Agrobacterium tumefaciens.
Figure 6: a, Stereoview of the backbone trace of a CENDH subunit (orange) together with the location of the 35 residues (yellow) conserved between the sequences of CENDH, DHLO and DHNO.

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Acknowledgements

We thank the support staff at the Synchrotron Radiation Source at CCLRC Daresbury Laboratory for assistance with station alignment. This work was supported by grants from The Wellcome Trust, EU, New Energy and Industrial Development Organisation and International Scientific Research (Joint Research), supported by The Ministry of Education, Science, Sports and Culture of Japan. We acknowledge an award of a travel grant under The British Council/The Royal Society/JSPS Anglo-Japanese Scientific Exchange Scheme. The Krebs Institute is a designated BBSRC Biomolecular Sciences Centre and a member of the North of England Structural Biology Centre (NESBIC).

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Authors and Affiliations

  1. Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, The University of Sheffield, Sheffield, S10 2TN, UK

    K.L. Britton & D.W. Rice

  2. Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama, 939-03, Japan

    Y. Asano

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Correspondence to K.L. Britton.

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Britton, K., Asano, Y. & Rice, D. Crystal structure and active site location of N-(1-D-carboxylethyl)-L-norvaline dehydrogenase. Nat Struct Mol Biol 5, 593–601 (1998). https://doi.org/10.1038/854

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