Aminolevulinate synthase: functionally important residues at a glycine loop, a putative pyridoxal phosphate cofactor-binding site

Gong, Jian; Ferreira, Glória C.

doi:10.1021/bi00005a024

Cited by 30 publications

(24 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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). In the threedimensional structures of these PLP-dependent enzymes, this glycine-rich loop associates with the phosphate group of PLP through hydrogen bonding and multiple van der Waals contacts.…”

Section: Discussionmentioning

confidence: 59%

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

View full text Add to dashboard Cite

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]...

show abstract

Section: Discussionmentioning

confidence: 59%

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

View full text Add to dashboard Cite

show abstract

“…In particular, a glycine-rich loop has been recognized at the co-factor binding site in those enzymes for which crystal structures are available and has also been identified through site-directed mutagenesis in threonine and D-serine dehydratases (Dana et al, 1987;Marceau et al, 1990) and more recently in aminolevulinate synthase (Gong et al, 1995(Gong et al, , 1996. Interestingly, a similar glycine-rich region is present in DDC (138-GEGGG; Maras et al, 1991), although its function has not been directly probed.…”

Section: Discussionmentioning

confidence: 99%

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

View full text Add to dashboard Cite

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kc,, values of about 50% and 15%, respectively, at pH 6.8, while the K, values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (LJ-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-1 1 ]-+Ala mutant displays a reduced affinity for pyridoxal 5"phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pK, and pKb for the groups involved in catalysis were determined for the wild-type and the C l l l A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 -+ 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-1 1 1 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur JBiochern 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.Keywords: active site; decarboxylase; pyridoxal 5"phosphate; site-directed mutagenesis The versatility of pyridoxal 5"phosphate (PLP) as a co-factor is demonstrated by the wide variety of reactions that enzymes dependent upon it catalyze. Although detailed catalytic mechanisms and information correlating structure and function have been reported for many PLP dependent enzymes, such information is conspicuously lacking for PLP dependent decarboxylases.Even though aromatic amino acid decarboxylases have been cloned from a number of mammalian (Ichinose et al., 1989;Tanaka et al., 1989;Kang & Joh, 1990;Taketoshi et al., 1990), insect (Eveleth et al., 1986, and plant sources (DeLuca et al., 1989;Kawalleck et al., 1993; Facchini & DeLuca, 1994)

show abstract

“…The strategy used for designing the library of constructs was similar to those previously described (28,29). Staggered oligomers with 15 complementary oligonucleotides were designed to span the wild-type mALAS2 cDNA sequence between the KpnI and AvrII restriction sites.…”

Section: Methodsmentioning

confidence: 99%

“…(See Ref. 28 for sequence of oligonucleotide B.) An annealing reaction, with a final volume of 6 l and containing 60 pmol of each oligonucleotide, was incubated at 65°C for 2 min and slowly cooled down to room temperature.…”

Section: Methodsmentioning

confidence: 99%

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

Stojanovski¹,

Ferreira

2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: 5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis. Results: N150H and N150F mutations significantly reduced the rate of quinonoid intermediate formation in the forward direction, while increasing the reverse reaction rate. Conclusion: Asn-150 is essential for establishing a catalytic balance between the forward and reverse reactions. Significance: This is the first report of an ALAS region modulating the ALA synthesis-breakdown balance.

show abstract

Aminolevulinate synthase: functionally important residues at a glycine loop, a putative pyridoxal phosphate cofactor-binding site

Cited by 30 publications

References 31 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

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

Contact Info

Product

Resources

About