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Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine

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Abstract

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO 2−4 ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 sub-sites in these three carboxypeptidases was revealed. It was found that SO 2−4 ion occupies the same position in the S1’ subsite as the C-terminal carboxy group of the substrate.

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Abbreviations

BOC-L-leucine:

N-(tert-butoxycarbonyl)-L-leucine

CABS-Sepharose:

p-aminobenzylsuccinyl-Sepharose 4B

CHAPS:

3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate

CPA:

carboxypeptidase A

CPB:

carboxypeptidase B

CPT:

carboxypeptidase T

DFP:

diisopropyl fluorophosphate

IPTG:

isopropyl-β-D-thiogalactopyranoside

MPD:

2-methyl-2,4-pentadiol

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Authors and Affiliations

  1. Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 119333, Moscow, Russia

    V. I. Timofeev, S. A. Kuznetsov & I. P. Kuranova

  2. Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 117545, Moscow, Russia

    V. Kh. Akparov & G. G. Chestukhina

Authors
  1. V. I. Timofeev
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  2. S. A. Kuznetsov
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  3. V. Kh. Akparov
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  4. G. G. Chestukhina
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  5. I. P. Kuranova
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Corresponding author

Correspondence to I. P. Kuranova.

Additional information

Original Russian Text © V. I. Timofeev, S. A. Kuznetsov, V. Kh. Akparov, G. G. Chestukhina, I. P. Kuranova, 2013, published in Biokhimiya, 2013, Vol. 78, No. 3, pp. 338–347.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM12-283, January 20, 2013.

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Timofeev, V.I., Kuznetsov, S.A., Akparov, V.K. et al. Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine. Biochemistry Moscow 78, 252–259 (2013). https://doi.org/10.1134/S0006297913030061

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  • DOI: https://doi.org/10.1134/S0006297913030061

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