A role for quaternary structure in the substrate specificity of leucine dehydrogenase

Baker, Patrick J.; Turnbull, A.P.; Sedelnikova, Svetlana E.; Stillman, Timothy J.; Rice, David W.

doi:10.1016/s0969-2126(01)00204-0

Cited by 92 publications

(98 citation statements)

References 41 publications

(47 reference statements)

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“…Results of structural studies in the last decade have revealed that these enzymes share a very similar subunit structure, with each subunit composed of two domains separated by a cleft harboring the active site [24][25][26]. To study the structure and function of the domains of amino acid dehydrogenases before their crystal structures were available, we constructed and characterized the chimeric enzyme consisting of an N-terminal domain of thermostable…”

Section: Methodsmentioning

confidence: 99%

“…On the basis of the molecular structures of GluDH [24], LeuDH [25], and PheDH [26], substrate specificity of amino acid dehydrogenases is explained by the types of residues comprising the substrate side-chain binding pocket. In GluDH of Clostridium symbiosum, the principal interactions that determine the specificity are between the γ-carboxyl group of the substrate glutamate and the amino group of K89 and the hydroxyl group of S380, with G90, A163, and V377 interacting with the hydrophobic component of the glutamate side chain [29].…”

Section: Methodsmentioning

confidence: 99%

“…In the Bacillus sphaericus LeuDH structure, the latter three residues are conserved (G41, A113, and V291, respectively), whereas K89 and S380 in GluDH are replaced by L40 and V294 in LeuDH (residue numbers refer to B. sphaericus LeuDH), making up a more hydrophobic substrate side-chain binding pocket [25]. X-ray crystallographic and homologybased molecular modeling studies of PheDH revealed that this enzyme has a hydrophobic and large side-chain binding pocket enough to bind the phenylalanine benzene ring by replacing glycine for A113 of LeuDH [18,30].…”

Section: Methodsmentioning

confidence: 99%

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Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Kataoka

Tanizawa

2003

Journal of Molecular Catalysis B: Enzymatic

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The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward L-phenylalanine and phenylpyruvate with 1.6 and 7.8% of k cat values of the wild-type enzyme for the preferred substrates, L-leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Kataoka

Tanizawa

2003

Journal of Molecular Catalysis B: Enzymatic

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“…These include the enzymes leucine dehydrogenase (LeuDH) (Nagata et al, 1988), glutamate dehydrogenase (GIuDH) , phenylalanine dehydrogenase (PheDH) and valine dehydrogenase (ValDH) (Turnbull et al, 1998;Leiser et al, 1996). The threedimensional structures of species variants of two members of this enzyme superfamily, the hexameric GIuDH and the octameric LeuDH (Baker et al, 1995) have been determined. While the quaternary structures of these two i" Present address: IRBM Istituto di Ricerche Biologia Molecolare P. Angeletti Via Pontina 00040 Pomezia-Roma, Italy.…”

Section: Introductionmentioning

confidence: 99%

Crystallization of NAD⁺-dependent phenylalanine dehydrogenase fromNocardia sp239

Pasquo

Britton

Baker

et al. 1998

Acta Crystallogr D Biol Cryst

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The NAD~-dependent phenylalanine dehydrogenase from Nocardia sp239 has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Two crystal forms were obtained in the presence and absence of the enzyme substrates phenylpyruvic acid or phenylalanine and its coenzyme NADH. Crystals of the native protein belong to the hexagonal system, with the space group being one of the enantiomorphic pair P6122 or P6522. The cell dimensions are a = b = 111.0, c = 174.5 A,, o~ = fl = 90 and F = 120 (>. Crystals grown from the protein co-crystallized with its substrates all belong to the trigonal system, space group P3 ~21 or P3221, with unit-cell dimensions of a = b = 88.1, c = 112.6A, ~ = fl = 90 and F = 120". Preliminary proteinsequencing experiments have established that this enzyme is related to the octameric PheDH's which are members of the wider superfamily of amino-acid dehydrogenases. However, gel-filtration studies suggest that this enzyme is active as a monomer. The full determination of the three-dimensional structure of this phenylalanine dehydrogenase will add to the understanding of the molecular basis of the differential substrate specificity within this enzyme superfamily. In turn this will contribute to the rational design of an amino-acid dehydrogenase which could be used for the diagnosis of phenylketonuria and for the chiral synthesis of high-value pharmaceuticals.

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“…Asparagine at this position has in fact been found so far only in B. sphaericus, and modeling suggests FIGURE 1: Sequence alignment of PheDHs using structural information from C. symbiosum GluDH and B. sphaericus LeuDH showing the residue (bold) that is thought to be an important determinant of phenylalanine/tyrosine specificity. Sequences shown are for GluDH from C. symbiosum (C. sym) (16), PheDH from B. sphaericus (B. sph) (27), PheDH from B. badius (B. bad) (17), PheDH from S. ureae (S. ure) (18), PheDH from T. intermedius (T. int) (28), and LeuDH from B. sphaericus (B. sph) (14). The secondary structure of C. symbiosum GluDH is indicated above the sequences and that of B. sphaericus LeuDH below.…”

Section: Resultsmentioning

confidence: 99%

Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates

et al. 2002

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Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K m values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K m for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K m values.The label phenylalanine dehydrogenase (PheDH 1 ) (EC 1.4.1.20) denotes a class of enzymes that catalyze the reversible NAD + -dependent oxidative deamination of a broad range of hydrophobic amino acid substrates, with particular preference for aromatic side chains. The enzyme from Bacillus sphaericus is unusual within this class since its activities toward L-phenylalanine and L-tyrosine are similar (1, 2). In contrast, PheDH from Rhodococcus sp. M4 shows 34-fold discrimination in favor of L-phenylalanine (3). Similarly, with the enzymes from Bacillus badius and Sporosarcina ureae, tyrosine gives only 9 and 5.4%, respectively, of the specific activity with phenylalanine (1, 4). It is of interest to understand the structural basis for this difference in discrimination between aromatic amino acid substrates that differ by only a single hydroxyl substituent. Determining the molecular details of substrate recognition in the family of amino acid dehydrogenases has also practical relevance, as these enzymes are useful as biocatalysts for stereospecific synthesis of amino acids, and as diagnostic reagents for a variety of inborn errors of amino acid metabolism. Indeed, the thermostability of PheDH from B. sphaericus would recommend its use as a diagnostic reagent. However, major interference from tyrosine makes it unsuitable for measuring serum L-phenylalanine levels in the diagnosis of phenylketonuria (PKU) (2).The rational design of enzymes with altered substrate specificities for use in chemical synthesis or clinical diagnosis is a major goal of our ...

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A role for quaternary structure in the substrate specificity of leucine dehydrogenase

Cited by 92 publications

References 41 publications

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Crystallization of NAD⁺-dependent phenylalanine dehydrogenase fromNocardia sp239

Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates

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A role for quaternary structure in the substrate specificity of leucine dehydrogenase

Cited by 92 publications

References 41 publications

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Crystallization of NAD+-dependent phenylalanine dehydrogenase fromNocardia sp239

Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates

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Crystallization of NAD⁺-dependent phenylalanine dehydrogenase fromNocardia sp239