The purpose of this research was to characterize the Escherichia coli, E. coli, allantoinase enzyme. Allantoinase catalyses the conversion allantoin to allantoate via the hydrolysis of a cyclic amide bond and is coded for by the allB gene. The enzyme is a member of the amidohydrolase superfamily. Amidohydrolase superfamily enzymes have a common (αβ)8-barrel structure but catalyze the hydrolysis of many different substrates by a common mechanism. The structural characteristics and roles of divalent cations of enzymes in this superfamily will be discussed and related to previous work conducted on allantoinases. In this work, the metal dependence of allantoinase was initially studied by Mn, Co, Zn, Cd, and Ni-supplemented assays of enzyme of very low metal content. By changing the growth conditions under which the allB was overexpressed in E. coli, and the addition of Zn, Co or Mn to the culture, enzyme with bound Zn (ZnALN), Co (CoALN) or Mn (MnALN) was produced. The pH dependence of log (kcat/KM) for allantoinase in the presence of MnCl2, ZnALN and CoALN followed a bell-shaped curve, indicating that one ionizable group needed to be deprotonated and the deprotonation of a second group caused a decrease in catalytic activity. The pK1 for ionization at low pH was dependent upon which divalent cation was present and is concluded to be that of the deprotonation of water. A structural model of allantoinase with bound allantoin was constructed and used to determine which amino acid residues may be involved in catalysis. Allantoinase mutants R67K, C152A, C152S, C287A, C287S, S317A, D315N and W332F were purified. The kinetic parameters kcat, KM and kcat/KM of wild type and mutant allantoinases were compared. The possible roles of these amino acid residues in catalysis and substrate binding, and the results of the pH rate profiles are discussed. A catalytic mechanism for allantoinase is proposed.

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>