| Alanine racemase (EC5.1.1.1) is a pyridoxal 5'- phosphate (PLP) dependent enzyme that catalyzes the interconversion of L- and D -alanine isomers. As the enzyme exists in a mostly prokaryotes (mostly in prokaryotes),such as B.subtilis, Salm onella typhim urium, Escherich ia coli,Streptococcus faeca lis,Pseudomonas and Mycobacterium,but generally absent in higher eukaryotes. Up to now, two alanine racemase, alr-encoded and dadX-Qncoded were identified in bacteria. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. The dadX-encoded alanine racemase is essential only for L-Ala catabolism. The alr-encoded alanine racemase which is constitutively expressed in the cells, plays a critical role in the bacterial growth by providing D-alanine, which is essential for peptidoglycan biosynthesis in the cell wall of gram-positive and gram-negative bacteria.In this paper, two predicted alanine racemase genes from the Pseudomonas putida YZ-26, termed Alr and dadX were isolated, Alr has an open reading frame (ORF) of 1230bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44.2kDa, and dadX has an open reading frame (ORF) of 1074bp, encoding a protein of 358 amino acids with a calculated molecular weight of 38.6 kDa. A homology comparison revealed identities of 94.4% to Pseudomonas putida KT2440 and 27.3%, 26.9%,27.4% of the Alr alanine racemase to Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, respectively. The amino acids sequence which deduced from dadX gene showed the homologies of 98.0%,71.1%,46.7% and 45.2% to those from Pseudomonas putida KT2440,Pseudomonas aeruginosa,Escherichia coli,Salmonella typhimurium, respectively.The two genes were then cloned into vector pETM3c and pMAL-s to form recombinant plasmid pETM3c-Alr and pMAL-s-dadX. Both recombinant protein were expressed in BL21 (DE3) and soluble. The recombinant alanine racemase was efficiently purified to homogeneity by Ni-Agarose column or Amylose column combination with Sephacryl-S-200 gel filtration. Alanine racemase activity was assayed by the spectrophotometric assay.The result were shown that dadX is only specific to L-alanine, while Alr is highly specific toL-alanine and L- isoleucine.The results revealed that:the kinetic properties of Alr is Km=10.34 mmol/L,Vmax=18.73μmol/min.mg; while in DadX, the Km=6.39 mmol/L and Vmax =8.83μmol/min.mg, respectively. The Alr shows the highest activity at pH 8.0 in the conversion, its optimum temperature is 37℃, and relatively stable in the pH range of 6.0-12.0, below the 40℃.On the contrary, dadX is highly specific to L-alanine at pH 7.0 in the conversion. and relatively stable in the pH range of 6.0-10.0.Both of enzymes were inhibited by 1mM Cu2+,lmM Zn2+,but activated by Sr2+,Mn2+,Co2+The initial crystallization trials were carried out at 16℃in 48-well plates with Crystal Screen kitsⅠ/Ⅱand PEG/ION kitsⅠ/Ⅱ(Hampton Research), and further optimization were tested with variation of precipitant concentration, pH and protein concentration.The crystal of Alr was obtained using the sitting-drop vapor diffusion technique in the presence of 0.2M ammonium sulfate and 0.1M sodium acetate trihydrate pH4.8,25%PEG 4000. The oblong shape crystal was used for data collection on a CCD detector on Rigaku R-AxisⅣ++) Cu Ka radiation (λ=1.5418 A) to 2.4 A°resolution at National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences.Structural studies on Alr from YZ-26 have revealed that Alr is a dimer, with each monomer consisting of two different domains:an eightstranded a/(3 barrel at the N terminus, and a C-terminal domain that is formed primarily withβ-strands. The alanine racemase monomer is composed of two domains, an eight-strandedα/βforming a TIM barrel at the N-terminus, which includes residues 50-270, which is the enzyme active site region, including the (β2β9 theβ-sheet stocks,α1～α9α-helix,and aβ-strand (residues 247-381) at theC-terminus. |
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