Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.     

A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution

We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain. The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57–59% aa identity with human DHPase, and 74–77% aa identity with each other. We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3. Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse. Human DRP-3 showed 94–100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons. Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39–42%) and Caenorhabditis elegans unc-33 (32–34%). Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells

The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0–6.5. The enzyme was stable against heat treatments between 4 and 50°C and works optimally at 52°C. The activity remained constant at 4°C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.     

Zinc hydrolases: the mechanisms of zinc-dependent deacetylases

A class of metalloenzymes, known as zinc hydrolases, catalyze a variety of hydrolytic reactions on many different substrates in important metabolic pathways. Deacetylation is an example of one of the types of reactions catalyzed by zinc hydrolases. The biological importance of the reactions catalyzed by many zinc hydrolases, including zinc-dependent deacetylases, has made these enzymes pharmaceutical targets for the development of inhibitors and, therefore, a clear understanding of the mechanisms of these enzymes is warranted. This review focuses on the current understanding of the mechanisms catalyzed by various zinc-dependent deacetylases and, in particular, the reaction mechanism catalyzed by the enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, also known as LpxC. In general, the zinc-water functions as the nucleophile with zinc stabilization of the tetrahedral intermediate and general-acid-base catalysis (GABC) provided by enzyme residue(s). Two types of GABC mechanisms have been identified, one that uses a single bifunctional GABC and another that uses a GABC pair.     

Aminolytic reaction catalyzed by D-stereospecific amidohydrolases from Streptomyces spp

From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe-d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe-d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30-60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity.     

The molecular basis of aminoacylase 1 deficiency

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.     

Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].