The Protein Journal

, Volume 26, Issue 6, pp 435–444

Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes

  • Maela León
  • Pablo Isorna
  • Margarita Menéndez
  • Juliana Sanz-Aparicio
  • Julio Polaina
Article

DOI: 10.1007/s10930-007-9083-2

Cite this article as:
León, M., Isorna, P., Menéndez, M. et al. Protein J (2007) 26: 435. doi:10.1007/s10930-007-9083-2

Abstract

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic β-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of β-strands arranged in supernumerary β-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary β-strands were introduced by site directed mutagenesis into the sequence encoding the β-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type β-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).

Keywords

Alpha-beta-barrelβ-glycosidasesPaenibacillus polymyxaSulfolobus solfataricus

Abbreviations

CD

Circular dichroism spectroscopy

IPTG

Isopropyl β-D-1-thiogalactopyranoside

UV

Ultraviolet

NMR

Nuclear magnetic resonance

PCR

Polymerase chain reaction

PNPG

p-Nitrophenyl-β-D-glucopyranoside

PDB

Protein data bank

TIM

Triose-phosphate isomerase

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Maela León
    • 1
  • Pablo Isorna
    • 2
  • Margarita Menéndez
    • 2
  • Juliana Sanz-Aparicio
    • 2
  • Julio Polaina
    • 1
  1. 1.Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones CientíficasBurjassotSpain
  2. 2.Instituto de Química-Física Rocasolano, CSICMadridSpain