Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans.
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.
Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC. wcwang@life.nthu.edu.tw
Wen-Ching
WC
Wang
Wei-Chun
WC
Chiu
Shih-Kuang
SK
Hsu
Chun-Li
CL
Wu
Cheng-Yu
CY
Chen
Jai-Shin
JS
Liu
Wen-Hwei
WH
Hsu
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D013379:Substrate Specificity
D000596:Amino Acids; D001426:Bacterial Proteins; D000591:Amino Acid Isomerases