A Novel Metal-activated Pyridoxal Enzyme with a Unique Primary Structure, Low Specificity d-Threonine Aldolase fromArthrobacter sp. Strain DK-38

Abstract

The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/Dallo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. strain DK-38. The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps. The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5-phosphate per mol of subunit, and Lys 59 of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH 4 . In addition, Mn 2؉ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions. Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn 2؉ ion per mol of subunit. Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5-phosphatedependent enzymes, indicating that low specificity Dthreonine aldolase is a new pyridoxal enzyme with a unique primary structure. Taken together, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme.The bioorganic synthesis of ␤-hydroxy-␣-amino acids attracts a great deal of attention because of their potential application as chiral building blocks for the synthesis of biologically active molecules (1-5). A variety of ␤-hydroxy-␣-amino acids is present in complex natural compounds with interesting biological properties. 3,4,5-Trihydroxy-L-aminopentanoic acid is a key component of polyoxins (1). 4-Hydroxy-L-threonine, for example, is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5Ј-phosphate (PLP) 1 -dependent enzymes (1). The D-isomers are also biologically significant, because they not only exist in mature mammals (6) but are also constituents of a range of antibiotics, for example, Fusaricidin (7) and Viscosin (8).Threonine aldolase (TA) (EC 4.1.2.5), which catalyzes the reversible interconversion of certain ␤-hydroxy-␣-amino acids and glycine plus aldehydes, has been shown to be useful for the biosynthesis of ␤-hydroxy-␣-amino acids (1-5). The enzyme appears to fall into two types, L-type and D-type, on the basis of its stereospecificity of the cleavage reaction toward the ␣-carbon of threonine. L-Type TA, acting on L-and/or L-allo-threonine, is further divided into three groups based on its stereospecificity toward the ␤-carbon of threonine as follows: (i) L-allo-TA is specific to L-allo-threonine; (ii) L-TA acts only on L-threonine; and (iii) low specificity L-TA can use both L-threonine and L-allo-threonine as substrates. All of the three L-type enzymes have been found to exist in nature. L-TA was partially purified from Clostridium pasteurian...

Direct Calcium Binding Results in Activation of Brain Serine Racemase

“…4). This spectral shift has been observed both in free (41,42) and enzyme-bound PLP (27,43) when reacting with L-cysteine and has been ascribed to the appearance of a stable complex containing a thiazolidine ring.…”

“…4A). However, since L-cysteine is also known to react with some pyridoxal phosphate enzymes (27), we inspected the spectral changes that occur in the visible region upon the addition of L-cysteine. As observed in Fig.…”

Crystal Structure of a Zinc-dependent d-Serine Dehydratase from Chicken Kidney

“…Our biochemical analysis showed that chDSD can use not only Zn 2ϩ but also Mn 2ϩ for the catalytic reaction, the latter ion being able to coordinate N and S atoms. Interestingly, D-threonine aldolases, which belong to fold-type III PLP-dependent enzymes, preserve the same metal-binding motif of His-X-Cys/Asp in the C-terminal region as found in chDSD and require an Mn 2ϩ ion for the ␣-elimination of D-threonine to produce acetoaldehyde and glycine (12). Although the crystal structure of D-threonine aldolases has not been solved, the present crystal structure of chDSD suggests that the hydroxyl group of the substrate D-threonine coordinates the Mn 2ϩ in the active site.…”

“…However, we have found recently that, in the chicken brain, D-serine is degraded mainly by a D-serine dehydratase (DSD) 4 (10), which catalyzes the ␣,␤-elimination of water from D-serine to form pyruvate and ammonia. In addition, we have found that the chicken DSD (chDSD) has a primary structure similar to those of metal-activated D-threonine aldolases (11)(12)(13), which are fold-type III PLP-dependent enzymes (14), and is distinct from a well known metal-independent bacterial DSD (dsdA) belonging to the foldtype II PLP-dependent enzyme family (14,15). Interestingly, a fold-type III family protein coded by gene YGL196W of Saccharomyces cerevisiae was recently identified as a zinc-dependent DSD (scDSD) (16).…”

Synthesis of 4-Hydroxyisoleucine by the Aldolase–Transaminase Coupling Reaction and Basic Characterization of the Aldolase fromArthrobacter simplexAKU 626

“…There have been some reports on PLPdependent aldolases. [13][14][15][16] It is not clear that the Arthrobacter aldolase requires PLP as a coenzyme, because the transaminase in the reaction mixture also requires PLP as a cofactor. Further characterization, cloning, and expression of the aldolase are in progress.…”