Functional Characterization of Alanine Racemase from
            <i>Schizosaccharomyces pombe</i>
            : a Eucaryotic Counterpart to Bacterial Alanine Racemase

Uo, Takuma; Yoshimura, Teizo; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

doi:10.1128/jb.183.7.2226-2233.2001

Cited by 67 publications

(46 citation statements)

References 37 publications

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“…ArgR is thought to be translocated via the Sec pathway by cleavage of an N-terminal signal peptide. According to the physiological role of alanine racemase [31] it is assumed that also amino acid racemase plays an important role for the synthesis of d-alanine and d-glutamate in the murein layer.…”

Section: Resultsmentioning

confidence: 99%

An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

Würges¹,

Mackfeld²,

Pohl³

et al. 2011

Adv Synth Catal

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We present an efficient method for the production of d-and l-allo-threonine (allo-Thr) with very high purity by enzymatic isomerization of l-or d-threonine (Thr) and simultaneous crystallization. Isomerization of Thr to allo-Thr is catalyzed by a purified amino acid racemase (AArac12996) from Pseudomonas putida NBRC12996, which can easily be obtained from a recombinant E. coli strain by secretion into the medium and subsequent anion exchange chromatography. Crystallization of d-and l-allo-Thr was performed in a repetitive batch mode over a period of up to 55 days at 30 8C. Total amounts of 30.8 g d-allo-Thr and 32.4 g l-allo-Thr were obtained with a very good diastereomeric excess of de d-allo > 99.2% and de l-allo > 98.4%, respectively, and in good yields (d-allo-Thr: 83%, l-alloThr: 79%). The enzymes remarkable high stability under process conditions resulted in enzyme specific yields of 2.56 g d-allo-Thr per mg AArac12996 and 1.62 g l-allo-Thr per mg AArac12996. In contrast to chemical multi-step syntheses of allo-Thr, our process consists of only one enzyme-catalyzed reaction step with simultaneous product crystallization. The process is performed under low energy consumption (30 8C, atmospheric pressure) in water and avoids the use and production of any toxic or harmful compounds. Recovery of the enantiomerically pure products is performed by simple filtration which reduces downstream processing significantly compared to chromatographic methods which are usually applied.

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Section: Resultsmentioning

confidence: 99%

An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

Würges¹,

Mackfeld²,

Pohl³

et al. 2011

Adv Synth Catal

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“…The enzyme occurs widely in bacteria and is essential in synthesis of peptidoglycan of bacterial cell walls by providing the D-enantiomer. There are some exceptions that some eukaryotes have the enzyme for biosynthesis of D-alanine-containing peptides in fungi (1,2), D-alanine metabolism in yeast (3), and osmotic regulation in a crayfish (4) and a bivalve mollusc (5). However, the peptidoglycan biosynthesis is unique to bacteria, and therefore the enzyme has been a target of antibacterial drugs (6 -10).…”

mentioning

confidence: 97%

Gene Cloning and Characterization of Alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei

Yokoigawa

Hirasawa

Ueno

et al. 2001

Biochemical and Biophysical Research Communications

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“…Alanine racemase is ubiquitous among bacteria, but is generally absent in higher eukaryotes with some exceptions: enzymes for D-alanine metabolism in yeast (7), the muscle of black tiger prawn (8) and alfalfa seedlings (9). Structure and properties such as amino acid sequence, quaternary structure, pH-dependency and kinetic parameters of eukaryotic enzymes seem to be distinct from those of bacterial alanine racemases.…”

mentioning

confidence: 99%

Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

Wen

et al. 2009

Journal of Bioscience and Bioengineering

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An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX OF4 ), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadX OF4 was purified to homogeneity by His 6 -tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40°C and the optimal pH was 10.5. The kinetic parameters K m and V max at 40°C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.

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Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe : a Eucaryotic Counterpart to Bacterial Alanine Racemase

Abstract: Schizosaccharomyces pombe has an open reading frame, which we named alr1؉ , encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1 ؉ gene in Escherichia coli and purified the gene product (

Cited by 67 publications

References 37 publications

An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

Gene Cloning and Characterization of Alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei

Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

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