Structure-based studies on the metal binding of two-metal-dependent sugar isomerases

Ponnandy Prabhu, TNT Doan, Manish Kumar Tiwari, Raushan Singh, Sun Chang Kim, Myoung-Ki Hong, Yun Chan Kang, Lin-Woo Kang, Jung-Kul Lee

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Two-metal-dependent sugar isomerases are important in the synthesis of rare sugars. Many of their properties, specifically their metal dependency, have not been sufficiently explored. Here we used X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and electron paramagnetic resonance spectroscopy to investigate the molecular determinants of the metal-binding affinity of L-rhamnose isomerase, a two-Mn2+-dependent isomerase from Bacillus halodurans (BHRI). The crystal structure of BHRI confirmed the presence of two metal ion-binding sites: a structural metal ion-binding site for substrate binding, and a catalytic metal ion-binding site that catalyzes a hydride shift. One conserved amino acid, W38, in wild-type BHRI was identified as a critical residue for structural Mn2+ binding and thus the catalytic efficiency of BHRI. This function of W38 was explored by replacing it with other amino acids. Substitution by Phe, His, Lys, Ile or Ala caused complete loss of catalytic activity. The role of W38 was further examined by analyzing the crystal structure of wild-type BHRI and two inactive mutants of BHRI (W38F and W38A) in complex with Mn2+. A structural comparison of the mutants and the wild-type revealed differences in their coordination of Mn2+, including changes in metal-ligand bond length and affinity for Mn2+. The role of W38 was further confirmed in another two-metal-dependent enzyme: xylose isomerase from Bacillus licheniformis. These data suggest that W38 stabilizes protein-metal complexes and in turn assists ligand binding during catalysis in two-metal-dependent isomerases.
Original languageEnglish
JournalFEBS Journal
Volume281
Issue number15
Pages (from-to)3446-3459
ISSN1742-464X
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • Amino Acid Substitution
  • Apoenzymes
  • Bacillus
  • Bacterial Proteins
  • Carbohydrate Epimerases
  • Catalytic Domain
  • Crystallography, X-Ray
  • Kinetics
  • Manganese
  • Models, Molecular
  • Protein Binding
  • Thermodynamics
  • 42Z2K6ZL8P Manganese
  • EC 5.1.3.- Carbohydrate Epimerases
  • binding site
  • catalytic efficiency
  • crystal structure
  • metal-binding affinity
  • substrate binding
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Endospore-forming Gram-Positives [07810] Bacillus licheniformis species Bacillus halodurans species
  • BHRI
  • L-rhamnose isomerase 9023-84-1 EC 5.3.1.14
  • manganese ion-dependent isomerase
  • xylose isomerase 9023-82-9 EC 5.3.1.5
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • Biochemistry and Molecular Biophysics
  • electron paramagnetic resonance spectroscopy laboratory techniques, spectrum analysis techniques
  • isothermal titration calorimetry laboratory techniques
  • site-directed mutagenesis laboratory techniques, genetic techniques
  • X-ray crystallography laboratory techniques, crystallographic techniques
  • 3P14 Protein Data Bank nucleotide sequence
  • 3UU0 Protein Data Bank nucleotide sequence
  • 3UVA Protein Data Bank nucleotide sequence
  • 3UXI Protein Data Bank nucleotide sequence
  • Enzymology
  • BIOCHEMISTRY
  • SITE-DIRECTED MUTAGENESIS
  • L-RHAMNOSE ISOMERASE
  • D-XYLOSE ISOMERASE
  • MEDIATED HYDRIDE SHIFT
  • ACTINOPLANES-MISSOURIENSIS
  • PSEUDOMONAS-STUTZERI
  • GLUCOSE ISOMERASE
  • ESCHERICHIA-COLI
  • X-RAY
  • SUBSTRATE-SPECIFICITY
  • Bacillus halodurans
  • isomerase
  • molecular determinant
  • rare sugar
  • METALS

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