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PubMed1997

N-arachidonylethanolamine (anandamide) formation from N-arachidonylphosphatidylethanolamine in rat brain membranes.

Labeled L-N-arachidonylphosphatidylethanolamine (L-N-arachidonyl PE), a likely precursor of N-arachidonylethanolamine (anandamide), as well as its D-isomer, were synthesized using [14C]arachidonic acid. Anandamide was formed by incubating L-N-arachidonyl PE and rat brain membrane with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of anandamide amidohydrolase. Formation of anandamide from L-N-arachidonyl PE was inhibited by p-chloromercuriphenylsulfonic acid (p-CMPS), sulfhydryl reagent, and heat inactivate pre-treatment. D-N-Arachidonyl PE, an unnatural analog for N-arachidonyl PE, did not form anandamide.

Animals↗

PubMed2002

Atrazine mineralization potential in two wetlands.

The fate of atrazine in agricultural soils has been studied extensively but attenuation in wetland systems has received relatively little attention. The purpose of this study was to evaluate the mineralization of atrazine in two wetlands in central Ohio. One was a constructed wetland, which is fed by Olentangy River water from an agricultural catchment area. The other was a natural fen (Cedar Bog) in proximity to atrazine-treated cornfields. Atrazine mineralization potential was measured by 14CO2 evolution from [U-ring-14C]-atrazine in biometers. The constructed wetland showed 70-80% mineralization of atrazine within 1 month. Samples of wetland water that were pre-concentrated 200-fold by centrifugation also mineralized 60-80% of the added atrazine. A high extent of atrazine mineralization (75-81% mineralized) was also associated with concentrated water samples from the Olentangy River that were collected upstream and downstream of the wetland. The highest levels of mineralization were localized to the top 5 cm zone of the wetland sediment, and the activity close to the outflow at the Olentangy wetland was approximately equal to that near the inflow. PCR amplification of DNA extracted from the wetland sediment samples showed no positive signals for the atzA gene (atrazine chlorohydrolase), while Southern blots of the amplified DNA showed positive bands in five of the six Olentangy wetland sediment samples. Amplification with the trzD (cyanuric acid amidohydrolase) primers showed a positive PCR signal for all Olentangy wetland sediment samples. There was little mineralization of atrazine in any of the Cedar Bog samples. DNA extracted from Cedar Bog samples did not yield PCR products, and the corresponding Southern hybridization signals were absent. The data show that sediment microbial communities in the Olentangy wetland mineralize atrazine. The level of activity may be related to the seasonality of atrazine runoff entering the wetland. Comparable activity was not observed in the Cedar Bog, perhaps because it does not directly receive agricultural runoff. Qualitatively, the detection of the genes was associated with measurable mineralization activity which was consistent with the differences between the two study sites.

Agriculture↗

PubMed2001

Effect of cell membrane of Agrobacterium radiobacter on enhancing D-amino acids production from racemic hydantoins.

An interesting phenomenon was observed that the existence of the intact cell membrane can enhance the D-amino acids production from D,L-5-substituted hydantoins by reacting with the whole cells of Agrobacterium radiobacter. Two intracellular enzymes were involved in the reaction process. The first enzyme D-hydantoinase converted hydantoins to carbamoyl derivatives which were further converted to D-amino acids by D-amidohydrolase. The amount of D-amino acids produced from hydantoins by the intact cells were 1.8-2.4 fold higher than the toluene treated cells. In addition, by using the intact cells the amount of D-amino acids produced from hydantoins was about 10 fold higher than that produced directly from carbamoyl derivatives. The relatively lower permeability of cell membrane to the reaction intermediate carbamoyl derivatives was confirmed by a simple mathematical model to be the main factor for the better performance of the intact cells for D-amino acid production. Besides, the low intracellular enzymes activities also contributed to the effect of intact cell membrane on enhancing the D-amino acid production.

Journal Article↗

PubMed2000

Dynamics of a mobile loop at the active site of Escherichia coli asparaginase.

Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover.

Asparaginase↗

PubMed1999

The effects of cannabinoids on the brain.

Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant Cannabis sativa is delta9-tetrahydrocannabinol (delta9-THC). In humans, psychoactive cannabinoids produce euphoria, enhancement of sensory perception, tachycardia, antinociception, difficulties in concentration and impairment of memory. The cognitive deficiencies seem to persist after withdrawal. The toxicity of marijuana has been underestimated for a long time, since recent findings revealed delta9-THC-induced cell death with shrinkage of neurons and DNA fragmentation in the hippocampus. The acute effects of cannabinoids as well as the development of tolerance are mediated by G protein-coupled cannabinoid receptors. The CB1 receptor and its splice variant CB1A, are found predominantly in the brain with highest densities in the hippocampus, cerebellum and striatum. The CB2 receptor is found predominantly in the spleen and in haemopoietic cells and has only 44% overall nucleotide sequence identity with the CB1 receptor. The existence of this receptor provided the molecular basis for the immunosuppressive actions of marijuana. The CB1 receptor mediates inhibition of adenylate cyclase, inhibition of N- and P/Q-type calcium channels, stimulation of potassium channels, and activation of mitogen-activated protein kinase. The CB2 receptor mediates inhibition of adenylate cyclase and activation of mitogen-activated protein kinase. The discovery of endogenous cannabinoid receptor ligands, anandamide (N-arachidonylethanolamine) and 2-arachidonylglycerol made the notion of a central cannabinoid neuromodulatory system plausible. Anandamide is released from neurons upon depolarization through a mechanism that requires calcium-dependent cleavage from a phospholipid precursor in neuronal membranes. The release of anandamide is followed by rapid uptake into the plasma and hydrolysis by fatty-acid amidohydrolase. The psychoactive cannabinoids increase the activity of dopaminergic neurons in the ventral tegmental area-mesolimbic pathway. Since these dopaminergic circuits are known to play a pivotal role in mediating the reinforcing (rewarding) effects of the most drugs of abuse, the enhanced dopaminergic drive elicited by the cannabinoids is thought to underlie the reinforcing and abuse properties of marijuana. Thus, cannabinoids share a final common neuronal action with other major drugs of abuse such as morphine, ethanol and nicotine in producing facilitation of the mesolimbic dopamine system.

Animals↗

PubMed2002

Impact of the biochemical assay for serum creatinine measurement on the individual carboplatin dosing: a prospective study.

We previously developed a formula to estimate the individual carboplatin clearance (CL) based on serum creatinine (Scr) determined by an enzymatic assay using creatinine amidohydrolase. An analytical comparison had shown systematic differences between this method and the commonly used Jaffé method (with Jaffé Scr (in microM)=1.08 x enzymatic Scr+1.6, as regression equation). We performed a pharmacokinetic prospective clinical study using the Jaffé assay to evaluate the impact of the method used for Scr measurement on the prediction of the carboplatin CL. In forty patients, carboplatin dosing was performed according to the Chatelut formula where the serum creatinine level was corrected according to the above equation. The population pharmacokinetics of carboplatin were analysed using the NONMEM program to determine the individual carboplatin CL from a limited sampling strategy. Thanks to the correction of the Jaffé Scr, no significant difference was observed between the administered and the optimal dose. In contrast, if no correction of the Scr was done, the patients would have been significantly under-dosed. Moreover, a covariate analysis using NONMEM gave a very consistent result showing that Scr should be decreased by 11.6% when the Jaffé value is used within the Chatelut equation. This study confirmed that differences in the Scr assay has consequences with regard to carboplatin dosing. The correction we propose for Scr obtained by the Jaffé method may help to standardise clinical practice.

Adult↗

PubMed2001

Structure of a human S-adenosylmethionine decarboxylase self-processing ester intermediate and mechanism of putrescine stimulation of processing as revealed by the H243A mutant.

S-Adenosylmethionine decarboxylase (AdoMetDC) is synthesized as a proenzyme that cleaves itself in a putrescine-stimulated reaction via an N-->O acyl shift and beta-elimination to produce an active enzyme with a catalytically essential pyruvoyl residue at the new N-terminus. N-->O acyl shifts initiate the self-processing of other proteins such as inteins and amidohydrolases, but their mechanisms in such proteins are not well understood. We have solved the crystal structure of the H243A mutant of AdoMetDC to 1.5 A resolution. The mutant protein is trapped in the ester form, providing clear evidence for the structure of the ester intermediate in the processing of pyruvoyl enzymes. In addition, a putrescine molecule is bound in a charged region within the beta-sandwich, and cross-links the two beta-sheets through hydrogen bonds to several acidic residues and ordered water molecules. The high-resolution structure provides insight into the mechanism for the self-processing reaction and provides evidence for the mechanism for simulation of the self-processing reaction by putrescine. Studies of the effects of putrescine or 4-aminobutanol on the processing of mutant AdoMetDC proenzymes are consistent with a model in which a single activator molecule interacts with buried Asp174, Glu178, and Glu256, leading to an alteration in the position of Glu11, resulting in stimulation of self-processing.

Adenosylmethionine Decarboxylase↗

PubMed2002

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

Amino Acid Sequence↗

PubMed2003

Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway.

PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 A crystal structure of native PhzD, and the 1.6 A structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.

Catalytic Domain↗

PubMed2003

High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli.

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.

Amino Acid Sequence↗

PubMed2005

Mechanism of the reaction catalyzed by isoaspartyl dipeptidase from Escherichia coli.

Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme have demonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end of a (beta/alpha)(8)-barrel domain. Steady-state kinetic parameters for the hydrolysis of beta-aspartyl dipeptides were obtained at pH 8.1. The pH-rate profiles for the hydrolysis of beta-Asp-Leu were obtained for the Zn/Zn-, Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for k(cat) and k(cat)/K(m) as a function of pH for all four metal-substituted forms. The pK(a) of the group that must be unprotonated for catalytic activity varied according to the specific metal ion bound in the active site, whereas the pK(a) of the group that must be protonated for catalytic activity was relatively independent of the specific metal ion present. The identity of the group that must be unprotonated for catalytic activity was consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. The identity of the group that must be protonated for activity was consistent with the free alpha-amino group of the dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residues Glu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutant enzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with beta-Asp-His, are consistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by the polarization of the amide bond via complexation to the beta-metal ion of the binuclear metal center. Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chain carboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond occur with donation of a proton from the protonated form of Asp285.

Asparagine↗

PubMed2006

Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction.

Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a widespread enzyme found in many bacterial species and all currently sequenced eukaryotic organisms. It occupies a key position at the branching point of two metabolic pathways: the tryptophan to quinolinate pathway and the bacterial 2-nitrobenzoic acid degradation pathway. The activity of ACMSD determines whether the metabolites in both pathways are converted to quinolinic acid for NAD biosynthesis or to acetyl-CoA for the citric acid cycle. Here we report the first high-resolution crystal structure of ACMSD from Pseudomonas fluorescens which validates our previous predictions that this enzyme is a member of the metal-dependent amidohydrolase superfamily of the (beta/alpha)(8) TIM barrel fold. The structure of the enzyme in its native form, determined at 1.65 A resolution, reveals the precise spatial arrangement of the active site metal center and identifies a potential substrate-binding pocket. The identity of the native active site metal was determined to be Zn. Also determined was the structure of the enzyme complexed with cobalt at 2.50 A resolution. The hydrogen bonding network around the metal center suggests that Arg51 and His228 may play important roles in catalysis. The metal center configuration of PfACMSD is very similar to that of Zn-dependent adenosine deaminase and Fe-dependent cytosine deaminase, suggesting that ACMSD may share certain similarities in its catalytic mechanism with these enzymes. These data enable us to propose possible catalytic mechanisms for ACMSD which appear to be unprecedented among all currently characterized decarboxylases.

Binding Sites↗

PubMed2006

Transition metal-catalyzed nonoxidative decarboxylation reactions.

Decarboxylases typically utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates. The recent characterization of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) has revealed that this enzyme adopts a TIM-barrel (beta/alpha)(8) fold and employs a mononuclear transition metal center to decarboxylate the substrate in an oxidant-independent fashion. Thus, ACMSD represents a type of decarboxylation reaction that has been so far uncharacterized in biological systems. Several close homologues of ACMSD were analyzed, including isoorotate decarboxylase (IDCase), 5-carboxyvanillic acid decarboxylase (5-CVD), gamma-resorcylate decarboxylase (gamma-RSD), and 4-oxalomesaconate hydratase (OMAH). These enzymes are involved in the catabolism of tryptophan and vanillate, the biodegradation of hydroxylbenzoates, and the thymidine salvage pathways in certain organisms. They possess the signature sequence motifs of the amidohydrolase superfamily and likely share the same structural and mechanistic characteristics as that of ACMSD. Analysis of the sequence conservation and evolutionary relationship of ACMSD-related proteins suggests an emerging ACMSD protein family that includes ACMSD and ACMSD-like decarboxylases and hydratases with diverse substrate specificities, many of which are poorly understood in regard to their functions and mechanisms. Progress in the biochemical and structural characterization of ACMSD not only sheds light on the active site of this protein family but also promises the elucidation of the detailed catalytic mechanism of these novel transition metal-dependent nonoxidative decarboxylation reactions.

Binding Sites↗

PubMed2006

Mechanistic characterization of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa catalyzes the deimination of N-formimino-l-glutamate in the histidine degradation pathway. An amino acid sequence alignment between HutF and members of the amidohydrolase superfamily containing mononuclear metal centers indicated that residues Glu-235, His-269, and Asp-320 are involved in substrate binding and activation of the nucleophilic water molecule. The purified enzyme contained up to one equivalent of zinc. The metal was removed by dialysis against the metal chelator dipicolinate with the complete loss of catalytic activity. Enzymatic activity was restored by incubation of the apoprotein with Zn2+, Cd2+, Ni2+, or Cu2+. The mutation of Glu-235, His-269, or Asp-320 resulted in the diminution of catalytic activity by two to six orders of magnitude. Bell-shaped profiles were observed for kcat and kcat/Km as a function of pH. The pKa of the group that must be unprotonated for catalytic activity was consistent with the ionization of His-269. This residue is proposed to function as a general base in the abstraction of a proton from the metal-bound water molecule. In the proposed catalytic mechanism, the reaction is initiated by the abstraction of a proton from the metal-bound water molecule by the side chain imidazole of His-269 to generate a tetrahedral intermediate of the substrate. The collapse of the tetrahedral intermediate commences with the abstraction of a second proton via the side chain carboxylate of Asp-320. The C-N bond of the substrate is subsequently cleaved with proton transfer from His-269 to form ammonia and the N-formyl product. The postulated role of the invariant Glu-235 is to ion pair with the positively charged formimino group of the substrate.

Bacterial Proteins↗

PubMed1996

Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

Aspartate Aminotransferases↗

PubMed1999

Xanthine degradation and related enzyme activities in leaves and fruits of two coffea species differing in caffeine catabolism.

The degradation of xanthine was studied in young and aged leaves and in immature and mature fruits of Coffea arabica and Coffea dewevrei, which differ with respect to caffeine catabolism. Radioisotope feeding experiments showed that leaves degraded xanthine more readily than fruits but that mature fruits and aged leaves were less efficient than younger tissues. In all cases, a significant part of the recovered radioactivity was in the ureides. Xanthine dehydrogenase was characterized as the enzyme responsible for xanthine degradation, and its activity and that of uricase were consistent with the results obtained in the radioisotope feeding experiments. Activities of allantoinase and allantoate amidohydrolase could not be detected. Considerable levels of endogenous allantoin and allantoic acid were found in fruits and leaves. Therefore, ureide accumulation might be a consequence of low enzyme activity. There was no positive correlation between urease activity and the data from the radioisotope feeding experiments.

Caffeine↗

PubMed2000

Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase.

[reaction--see text] Penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) was immobilized in a simple and effective way by physical aggregation of the enzyme, using a precipitant, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). These had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less. Penicillin G acylase CLEAs also catalyzed the synthesis of ampicillin in a broad range of organic solvents.

Ampicillin↗

PubMed2001

Pyrimidine base catabolism in Pseudomonas putida biotype B.

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.

Carbon↗