Structural Analysis of a Ternary Complex of Allantoate Amidohydrolase from Escherichia coli Reveals its Mechanics

Biology Department, Brookhaven National Laboratory, New York, New York, United States
Journal of Molecular Biology (Impact Factor: 4.33). 05/2007; 368(2):450-63. DOI: 10.1016/j.jmb.2007.02.028
Source: PubMed


Purine metabolism plays a major role in regulating the availability of purine nucleotides destined for nucleic acid synthesis. Allantoate amidohydrolase catalyzes the conversion of allantoate to (S)-ureidoglycolate, one of the crucial alternate steps in purine metabolism. The crystal structure of a ternary complex of allantoate amidohydrolase with its substrate allantoate and an allosteric effector, a sulfate ion, from Escherichia coli was determined to understand better the catalytic mechanism and substrate specificity. The 2.25 A resolution X-ray structure reveals an alpha/beta scaffold akin to zinc exopeptidases of the peptidase M20 family and lacks the (beta/alpha)(8)-barrel fold characteristic of the amidohydrolases. Arrangement of the substrate and the two co-catalytic zinc ions at the active site governs catalytic specificity for hydrolysis of N-carbamyl versus the peptide bond in exopeptidases. In its crystalline form, allantoate amidohydrolase adopts a relatively open conformation. However, structural analysis reveals the possibility of a significant movement of domains via rotation about two hinge regions upon allosteric effector and substrate binding resulting in a closed catalytically competent conformation by bringing the substrate allantoate closer to co-catalytic zinc ions. Two cis-prolyl peptide bonds found on either side of the dimerization domain in close proximity to the substrate and ligand-binding sites may be involved in protein folding and in preserving the integrity of the catalytic site.

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    • "This may indicate that differences in AAH enzymatic activity due to Mn2+ availability are responsible for the differences in shoot ureide concentration during water-deficit stress. The AAH enzyme is proposed to have a multimeric structure (Agarwall et al., 2007; Werner et al., 2008). The role of Mn2+ in enzyme assembly is unknown. "
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    ABSTRACT: Drought is a limiting factor for N(2) fixation in soybean [Glycine max (L.) Merr.] thereby resulting in reduced biomass accumulation and yield. Drought-sensitive genotypes accumulate ureides, a product of N(2) fixation, during drought stress; however, drought-tolerant genotypes have lower shoot ureide concentrations, which appear to alleviate drought stress on N(2) fixation. A key enzyme involved in ureide breakdown in shoots is allantoate amidohydrolase (AAH). It is hypothesized that AAH gene expression in soybean determines shoot ureide concentrations during water-deficit stress and is responsible for the differential sensitivities of the N(2)-fixation response to drought among soybean genotypes. The objectives were to examine the relationship between AAH transcript levels and shoot ureide concentration and drought tolerance. Drought-tolerant (Jackson) and drought-sensitive (Williams) genotypes were subjected to three water-availability treatments: well-watered control, moderate water-deficit stress, and severe water-deficit stress. Shoot ureide concentrations were examined, in addition to gene expression of AAH and DREB2, a gene expressed during water-deficit stress. As expected, DREB2 expression was detected only during severe water-deficit stress, and shoot ureide concentrations were greatest in the drought-sensitive genotype relative to the drought-tolerant genotype during water-deficit stress. However, expression of AAH transcripts was similar among water treatments and genotypes, indicating that AAH mRNA was not closely associated with drought tolerance. Ureide concentrations in shoots were weakly associated with AAH mRNA levels. These results indicate that AAH expression is probably not associated with the increased ureide catabolism observed in drought-tolerant genotypes, such as Jackson. Further study of AAH at the post-translational and enzymatic levels is warranted in order to dissect the potential role of this gene in drought tolerance.
    Journal of Experimental Botany 02/2009; 60(3):847-51. DOI:10.1093/jxb/ern332 · 5.53 Impact Factor
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    ABSTRACT: Soybean (Glycine max L. Merr.) establishes symbiosis with N 2 -fixing bacteria of the Rhizobia group, such as Bradyrhizobium sp. Specific molecules secreted by Bradyrhizobium, named nodulation factors, play a pivotal role in the development of root nodule. Inside nodules, rhizobia are differentiated into bacteroids, which reduce atmospheric nitrogen into ammonia. The major part of ammonia is assimilated into glutamine, which participates indirectly in nodule ureide synthesis. Among the leguminous family, soybean is one of the most sensitive to drought stress, which leads to a significant decrease in the biological nitrogen fixation (BNF). Drought-sensitive soybean genotypes accumulate ureides during drought stress; however, drought-tolerant genotypes have lower shoot ureide concentrations, which seem to alleviate drought stress on BNF. Researches based on new tools to increase BNF have been a priority during the last decade. Manganese fertilization under moderate drought conditions increases the catabolism of ureides and N 2 fixation in soybean. The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium cleaves ACC, the immediate precursor of ethylene in plants, decreasing the inhibitory effect of ethylene on nodulation. Induction of nodulation genes in Bradyrhizobium has positive effects on soybean growth under moderate drought stress. The aim of this review is focused to enclose new molecular targets that allow improving BFN in soybean under drought stress conditions. RESUMEN. La soya (Glycine max L. Merr.) establece simbiosis con bacterias fijadoras del nitrógeno de la familia de los rizobios; específicamente con bacterias del género Bradyrhizobium. Los rizobios secretan moléculas específicas denominadas factores Nod, que juegan un papel importante en el desarrollo del nódulo radicular. En el nódulo, los rizobios son diferenciados en bacteroides, donde ocurre la fijación biológica del nitrógeno (BNF) y se produce amonio. La mayor parte del amonio es asimilado en glutamina, que participa indirectamente en la síntesis de ureidos. La soya se considera una de las plantas leguminosas más sensibles al estrés por sequía, con una disminución significativa en la BNF. Los ureidos se acumulan en plantas de soya sensibles a la sequía durante el déficit hídrico, mientras que las plantas tolerantes presentan bajas concentraciones de ureidos que pueden reducir el estrés sobre la BNF. Se han realizado investigaciones dirigidas a incrementar la BNF en condiciones de estrés por sequía. La fertilización con manganeso en condiciones moderadas de déficit hídrico incrementa la degradación de los ureidos y la BNF. La enzima ACC desaminasa en los rizobios degrada el ACC, precursor inmediato del etileno en las plantas, y disminuye los efectos inhibitorios del etileno en la nodulación. La inducción de los genes de la nodulación en Bradyrhizobium sp. ha mostrado efectos positivos en el crecimiento de la soya en condiciones moderadas de sequía. El objetivo de esta revisión bibliográfica está dirigido a relacionar nuevos blancos moleculares que permitan incrementar la BNF en condiciones de estrés por sequía.
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    ABSTRACT: Enzymatic kinetic resolution is a widely used biotechnological tool for the production of enantiomerically pure/enriched compounds. This technique takes advantage of the enantioselectivity or enantiospecificity of an enzyme for one of the enantiomers of a racemic substrate to isolate the desired isomer. N-Carbamoyl-d- and l-amino acid amidohydrolases (d- and l-carbamoylases) are model enzymes for this procedure due to their strict enantiospecificity. Carbamoylase-based kinetic resolution of amino acids has been applied for the last three decades, allowing the production of optically pure d- or l-amino acids. Furthermore, this enzyme has become crucial in the industrially used multienzymatic system known as “Hydantoinase Process,” where the kinetic resolution produced by coupling an enantioselective hydantoinase and the enantiospecific carbamoylase is enhanced by the enzymatic/chemical dynamic kinetic resolution of the low-rate hydrolyzed substrate. This review outlines the properties of d- and l-carbamoylases, emphasizing their biochemical/structural characteristics and their biotechnological applications. It also pinpoints new applications for the exploitation of carbamoylases over the forthcoming years.
    Applied Microbiology and Biotechnology 01/2010; 85(3):441-458. DOI:10.1007/s00253-009-2250-y · 3.34 Impact Factor
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