Publications

• Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5'-phosphate-dependent enzymes.

  Kaeck H., Sandmark J., Gibson K., Schneider G., Lindqvist Y.

  The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a cr ... >> More

  The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified. << Less

  J. Mol. Biol. 291:857-876(1999) [PubMed] [EuropePMC]

• Purification and properties of 7, 8-diaminopelargonic acid aminotransferase. An enzyme in the biotin biosynthetic pathway.

  Stoner G.L., Eisenberg M.A.

  The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a ... >> More

  The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homogeneous by the scanning of of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values. The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incubation with the amino donor, S-adenosyl-L-methionine. A molecular weight estimation of 94,000 plus or minus 10, 000 has been obtained by gel filtration and sucrose gradient sedimentation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47, 000 plus or minus 3, 000 indicating a dimeric enzyme. A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equivalent to the 7, 8-diaminopelargonic acid produced in the reaction mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5'-methylthioadenosine and the neutral compound, 2-oxo-3-butenoic acid. << Less

  J. Biol. Chem. 250:4029-4036(1975) [PubMed] [EuropePMC]

• The dual-specific active site of 7,8-diaminopelargonic acid synthase and the effect of the R391A mutation.

  Eliot A.C., Sandmark J., Schneider G., Kirsch J.F.

  7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesi ... >> More

  7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesis of biotin. The wild-type enzyme has a steady-state kcat value of 0.013 s(-1), and the K(m) values for SAM and KAPA are 150 and <2 microM, respectively. The k(max) and apparent K(m) values for the half-reaction of the PLP form of the enzyme with SAM are 0.016 s(-1) and 300 microM, respectively, while those for the reaction with DAPA are 0.79 s(-1) and 1 microM. The R391A mutant enzyme exhibits near wild-type kinetic parameters in the reaction with SAM, while the apparent K(m) for DAPA is increased 180-fold. The 2.1 A crystal structure of the R391A mutant enzyme shows that the mutation does not significantly alter the structure. These results indicate that the conserved arginine residue is not required for binding the alpha-amino acid SAM, but it is important for recognition of DAPA. << Less

  Biochemistry 41:12582-12589(2002) [PubMed] [EuropePMC]

• Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis.

  Sandmark J., Eliot A.C., Famm K., Schneider G., Kirsch J.F.

  The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutati ... >> More

  The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor. << Less

  Biochemistry 43:1213-1222(2004) [PubMed] [EuropePMC]