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Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae
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 Title & Authors
Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae
Kang, Han-Chul; Yoon, Sang-Hong; Lee, Chang-Muk; Koo, Bon-Sung;
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 Abstract
A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.
 Keywords
alanine racemase;characteristics;Xanthomonas oryzae;
 Language
English
 Cited by
 References
1.
Badet B and Walsh C (1985) Purification of an alanine racemase from Streptococcus faecalis and analysis of its inactivation by (1-aminoethyl) phosphonic acid enantiomers. Biochemistry 24, 1333-1341. crossref(new window)

2.
Bradford MM (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72, 248-254. crossref(new window)

3.
Buell MV and Hansen RE (1960) Reaction of pyridoxal 5 phosphate with aminothiols. J Am Chem Soc 82, 6042-6049. crossref(new window)

4.
Copie V, Faraci WS, and Walsh CT (1988) Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'phosphate in the enzyme-inhibitor complex by solidstate N15-nuclear magnetic resonance. Biochemistry 27, 4966-4970. crossref(new window)

5.
Erion MD and Walsh CT (1987) 1-Aminocyclopropane-phosphonate: Time-dependen inactivation of 1-aminocyclopropanecarboxylate deaminase and Bacillus stearothermophilus alanine racemase by slow dissociation behavior. Biochemistry 26, 3417-3425. crossref(new window)

6.
Faraci WS and Walsh CT (1989) Mechanism of inactivation of alanine racemase by ${\beta},{\beta},{\beta}-trifluoroalanine$. Biochemistry 28, 431-437. crossref(new window)

7.
Francois JA and Kappock TJ (2007) Alanine racemase from the acidophile Acetobacter aceti. Prot Exp Purif 51, 39-48. crossref(new window)

8.
Hoffmann K, Schneider-Scherzer E, Kleinkauf H, and Zocher R (1994) Purification and characterization of eukaryotic alanine racemase acting as key enzyme in cyclosporine biosynthesis. J Biol Chem 269, 12710-12714.

9.
Johnston M (1969) Studies on amino acid racemases. J Biol Chem 244, 5414-5420.

10.
Ju J, Yokoigawa K, Misono H, and Ohnishi K (2005) Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli. J Biosci Bioeng 100, 409-417. crossref(new window)

11.
Kanodia S, Agarwall S, Singh P, Singh P, and Bhatnagar R (2008) Biochemical characterization of alanine racemase; a spore protein produced by Bacillus anthracis. Biochem Mol Biol Rep 42, 47-52.

12.
Kuniki K, Masaru S, and Yoneyama M (2007) Synthesis of DLtryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996. Appl Microbiol Biotechnol 73, 1299-1305. crossref(new window)

13.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. crossref(new window)

14.
Lambert MP and Neuhaus FC (1972) Mechanism of D-cycloserine action: Alanine racemase from Escherichia coli W. J Bacteriol 110, 978-987.

15.
Liardon R and Hurrell RF (1983) Amino acid racemization in heated and alkali-treated proteins. J Agric Food Chem 31, 432-437. crossref(new window)

16.
Lobocka M, Hennig J, and Wild J (1994) Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. J Bacteriol 176, 1500-1510.

17.
Mackay D (1962) The mechanism of the reaction of cysteine with pyridoxal 5-phosphate. Arch Biochem Biophys 99, 93-100. crossref(new window)

18.
Nishimura K, Tomoda Y, Nakamoto Y, Ishii Y, and Nagata Y (2007) Alanine racemase from the green alga Chlamydomonas reinhardtii. Amino acids 32, 59-62. crossref(new window)

19.
Noda M, Matoba Y, Kumagai T, and Sugiyama M (2004) Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product. J Biol Chem 279, 46153-46161. crossref(new window)

20.
Nomura T, Yamamoto I, Morishita F, Furukawa Y, and Matsushima O (2001) Purification and some properties of alanine racemase from a bivalve mollusk Corbicula japonica. J Exp Zool 289, 1-9. crossref(new window)

21.
Oikawa T, Tauch A, Schaffer S, and Fujioka T (2006) Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase. J Biotechnol 125, 503-512. crossref(new window)

22.
Panizzutti R, Miranda JD, Ribeiro CS, Engelender S, and Wolosker H (2001) A new strategy to decrease N-methyl-D-aspartate (NMDA) receptor coactivation: Inhibition of D-serine synthesis by converting serine racemase into an eliminase. Proc Natl Acad Sci USA 98, 5294-5299. crossref(new window)

23.
Preston RA and Douthit HA (1984) Germination of Bacillus cereus spores: Critical control by DL-alanine racemase. J Gen Microbiol 130, 3123-3133.

24.
Saito M, Nishimura K, Hasegawa Y, Shinohara T, Wakabayashi S, Kurihara T, Ishizuka M, and Nagata Y (2007) Alanine racemase from Helicobacter pylori NCTC11637. Life Sci 80, 788-794. crossref(new window)

25.
Sambrook J and Russel DW (2001) In Molecular Cloning: A Laboratory Manual, (3rd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

26.
Shibata K, Shirasuna K, and Motegi K (2000) Purification and properties of alanine racemase crayfish Procambarus clarkia. Comp Biochem Physiol 126, 599-608. crossref(new window)

27.
Silverman RB (1988) The potential use of mechanism based enzyme inactivators. J Enz Inhib 2, 73-90. crossref(new window)

28.
Strych U, Huang HC, Krause KL, and Benedik MJ (2000) Characterization of the alanine racemase from Pseudomonas aeruginosa PAO1. Curr Microbiol 41, 290-294. crossref(new window)

29.
Uo T, Ueda M, Nishiyama T, Yoshimura T, and Esaki N (2001b) Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon. J Mol Cat 137-144.

30.
Uo T, Yoshimura T, Tanaka N, Takegawa K, and Esaki N (2001a) Functional characterization of alanine racemase from Schizosaccharomyces pombe: A eukaryotic counterpart to bacterial alanine racemase. J Bacteriol 183, 2226-2233. crossref(new window)

31.
Wang E and Walsh CT (1978) Suicide substrates for the alanine racemase of Escherichia Coli. Biochemistry 17, 1313-1321. crossref(new window)

32.
Yamashita T, Ashiuchi M, Ohnishi K, Kato S, Nagata S, and Misono H (2003) Molecular characterization of alanine racemase from Bifidobacterium bifidum. J Mol Cat B 23, 213-222. crossref(new window)

33.
Zhang X, Jantama K, Moore JC, and Shanmugam KT (2007) Production of L-alanine by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 77, 355-366. crossref(new window)