Crystal Structure of a D-Amino Acid Aminotransferase: How the Protein Controls Stereoselectivity

Sugio, Shigetoshi; Petsko, Gregory A.; Manning, James M.; Soda, Kenji; Ringe, Dagmar

doi:10.1021/bi00030a002

Cited by 163 publications

(158 citation statements)

References 29 publications

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“…If Lys337 proves not to be part of the SAM binding domain, it may form an internal aldimine with PLP, which may be displaced by the substrate during catalysis. Such a mechanism has been reported for PLP-dependent amino acid aminotransferases (41). It has also been reported that a glycine-rich conserved region (GXXGXXGG) has been found in all known aminolevulinate synthases, glycogen phosphorylase b, and tryptophan synthase (17,21,29).…”

Section: Discussionmentioning

confidence: 54%

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

2000

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Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-␤-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. KAM from Clostridium subterminale SB4 catalyzes the interconversion of L-lysine and L-␤-lysine. In C. subterminale SB4, the production of ␤-lysine is the first step in the metabolism of lysine as the sole source of carbon and nitrogen, leading to acetate and butyrate as final products (39). Unlike other aminomutases, including D-lysine 5,6-aminomutase (1, 26), D-ornithine 2,3-aminomutase (2), and L-leucine 2,3-aminomutase (33), KAM is not adenosylcobalamin dependent. Instead, the enzyme is activated by SAM and contains iron-sulfur clusters and PLP (7,31,38).The interconversion of L-␣-lysine and L-␤-lysine is a reversible process in which an unactivated, carbon-bound hydrogen atom undergoes a 1,2-migration concomitant with the countermigration of the ␣-amino group by a novel free-radicalbased mechanism (12, 13). According to the current working hypothesis, homolytic cleavage of the S-adenosyl bond in SAM is mediated by the [4Fe-4S] center and produces the 5Ј-deoxyadenosyl radical as a transient intermediate (27,28). As illustrated in Fig. 1, the 5Ј-deoxyadenosyl radical initiates the rearrangement by abstracting the 3-pro-R hydrogen of L-lysine in the forward direction to form radical 1 or the 2-pro-R hydrogen of L-␤-lysine in the reverse direction to form radical 3.Evidence suggests that both amino acids are bound to the enzyme in the form of external aldimines with PLP. The substrate-and product-related free radicals are interconverted by way of an azacyclopropylcarbinyl, radical intermediate 2.KAM was first observed by Costilow and coworkers in crude extracts of C. subterminale SB4 (8) and later was purified and found to contain PLP and iron and to require SAM for activity (7). Enzyme activity was rapidly destroyed by dioxygen. The purified enzyme was partially inactive and could be further activated by prolonged anaerobic incubation with PLP, iron, and glutathione or dihydrolipoate, followed by addition of SAM and dithionite (7). KAM is a multisubunit protein with a subunit M r of 48,000 and an overall M r of 285,000 (7,38). Later studies demonstrated the presence of labile sulfide in the form of three [4Fe-4S]...

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Section: Discussionmentioning

confidence: 54%

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

2000

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“…It is, therefore, crucial for the enzyme to have a correct arrangement of the active site groups in order to accumulate E-Q. Although the stereospecificity of D-AAT is opposite to that of aspartate aminotransferase, crystallographic data revealed that the arrangement of the active site groups is quite similar (Sugio et al, 1995). The fact that the reaction of D-AAT with D-serine and erythro-β-hydroxy-DL-aspartate results in the accumulation of E-Q as a major peak, but the reaction of Dthreonine that has a threo β-hydroxy group could not, suggests the requirement of a certain structural arrangement of the active site groups for the accumulation of E-Q (Martinez del Pozo et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Effects of Salts on the Conformation and Catalytic Properties of D-Amino Acid Aminotransferase

Ro¹

2002

BMB Reports

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The effects of salts on the biochemical properties of Damino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UVvisible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with α-[ 2 H]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of saltbinding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of Damino acid aminotransferase.

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“…aspartate aminotransferase [5], o-amino acid:pyruvate aminotransferase [6], tyrosine phenol-lyase [7], and dialkylglycine decarboxylase [8], belong to the a-family and resemble each other very strongly in their three-dimensional structures [9]. o-Amino acid aminotransferase, which is only very distantly related to the members of the a-family, has a structural organisation completely different from those of any of the enzymes that utilize PLP [10]. Furthermore, the fl-subunit of tryptophan synthase, which catalyzes the PLP-dependent part of the reaction, is a member of the fl-family and has a tertiary structure [11] that bears no resemblance to that of the enzymes of the a-family.…”

Section: Introductionmentioning

confidence: 99%

Cloning, purification, and crystallization of Escherichia coli cystathionine β‐lyase

Laber¹,

Clausen

Huber

et al. 1996

FEBS Letters

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The metC gene coding for cystathionine ~-lyase of Escherichia coli has been cloned and used to construct an overproducing E. coli strain. An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent. The crystals belong to the orthorombic space group C2221. Their unit cell parameters are a = 60.9 A, b = 154.7 A and c = 152.7 ~,. Consideration of the possible values of VM accounts for the presence of one dimer per asymmetric unit. The crystals are suitable for X-ray analysis and a complete native date set to 1.83 ~, resolution has been collected using synchrotron radiation.

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Crystal Structure of a D-Amino Acid Aminotransferase: How the Protein Controls Stereoselectivity

Cited by 163 publications

References 29 publications

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

Effects of Salts on the Conformation and Catalytic Properties of D-Amino Acid Aminotransferase

Cloning, purification, and crystallization of Escherichia coli cystathionine β‐lyase

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