Publications

• Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

  Bisht S., Rajaram V., Bharath S.R., Kalyani J.N., Khan F., Rao A.N., Savithri H.S., Murthy M.R.

  Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruva ... >> More

  Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested. << Less

  J. Biol. Chem. 287:20369-20381(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Diaminopropionate ammonia-lyase from Salmonella typhimurium. Purification and characterization of the crystalline enzyme, and sequence determination of the pyridoxal 5'-phosphate binding peptide.

  Nagasawa T., Tanizawa K., Satoda T., Yamada H.

  We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native ... >> More

  We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5'-phosphate(pyridoxal-P)/mol of enzyme. The enzyme catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyridoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme and other pyridoxal-P-dependent enzymes. << Less

  J. Biol. Chem. 263:958-964(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.