Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans

J Mol Biol. 2004 Sep 3;342(1):155-69. doi: 10.1016/j.jmb.2004.07.023.

Abstract

N-acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure alpha-amino acids, a process that has potential industrial applications. Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans. The expressed NAAAR racemized various substrates at an optimal temperature of 60 degrees C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-D-methionine and N-acetyl-L-methionine, respectively. The crystal structure of NAAAR was solved to 1.3 A resolution using multiwavelength anomalous dispersion (MAD) methods. The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (beta/alpha)7 beta barrel domain. The NAAAR.Mg2+ and NAAAR.N-acetyl-L-glutamine.Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids. Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region. The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract alpha-protons of various carboxylate substrates efficiently. The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Isomerases / chemistry*
  • Amino Acid Isomerases / genetics
  • Amino Acid Isomerases / metabolism
  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • Deinococcus / enzymology*
  • Deinococcus / genetics
  • Models, Molecular
  • Molecular Conformation*
  • Molecular Sequence Data
  • Molecular Structure
  • Protein Structure, Quaternary*
  • Sequence Alignment
  • Substrate Specificity

Substances

  • Amino Acids
  • Bacterial Proteins
  • Amino Acid Isomerases

Associated data

  • PDB/1R0M