Properties of the hydantoinase from agrobacterium SP. IP I-671

Summary A hydantoin-hydrolyzing enzyme has been purified from an newly isolatedAgrobacterium sp. by procedures including ammonium sulfate fractionation and ion-exchange chromatography. Kinetic studies have demonstrated that this enzyme, which is strictlyd-selective and has a broad substrate specificity exhibits remarkable stability. Microbial bioconversion at 60°C and pH 10.0, allowed complete conversion of 30 g/L ofd,l 5-benzylhydantoin into thed N-carbamyl derivative of phenylalanine (molar yield of 96%) in less than 10 h. The hydantoinase is activated by Ni²⁺ ions.     

Mutational analysis of the hydantoin hydrolysis pathway in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> RU-KM3<Subscript>S</Subscript>

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding d-amino acids or l-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase (N-carbamoylase). Pseudomonas putida strain RU-KM3_S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and l-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and -ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not -ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or -ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3_S cells.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

Efficient production of the industrial biocatalysts hydantoinase and N-carbamyl amino acid amidohydrolase: Novel non-metabolizable inducers

Abstract The biosynthesis of the hydantoin-hydrolysing enzymes hydantoinase and N -carbamyl amino acid amidohydrolase from Agrobacterium sp. IP I-671, a Gram-negative bacterium used as a biocatalyst for the production of enantiomerically pure ( R ) amino acids, was found to be highly inducible by the addition to the cultivation medium of different non-metabolizable thiolated hydantoins or pyrimidines. Among these inducers the hexacyclic pyrimidine thioderivatives were more potent than all the pentacyclic thiohydantoin compounds. Addition of 2,4-thiouracil to the cultures, at a rate of 0.1 g (g cell dry mass)⁻¹, led to no appreciable growth inhibition and yielded a biocatalyst exhibiting a 40-fold higher hydantoinase and a 15-fold higher N -carbamyl amino acid amidohydrolase activity than the corresponding inducer-free cultures.     

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

Structure and Function of Amino Acid Ammonia-lyases

Histidine ammonia-lyase (HAL) and methylaspartate ammonia-lyase (MAL) belong to the family of carbon-nitrogen lyases (EC 4.3.1). The enzymes catalyze the α,β-elimination of ammonia from (S)-His to yield urocanic acid, and (S)-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid, respectively. Based on structural analyses, the peptide at the active center of HAL from Pseudomonas putida is considered to be post-translationally dehydrated to form an electrophilic 4-methylidene-imidazole-one (MIO) group. A reaction mechanism was proposed with the structure. On the other hand, the structure of MAL from Citrobacter amalonaticus was found to be a typical TIM barrel structure with Mg²⁺ coordinated to the 4-carbonyl of the substrate methylaspartate. Unlike HAL, MIO was not observed in MAL, and the reaction of MAL appears to be completely different from phenylalanine ammonia-lyase (PAL), HAL, and other amino acid ammonia-lyases. A reaction mechanism is proposed in which the hydrogen at the β to the amino group of the substrate is abstracted forming an enolate type intermediate and then ammonia is released.     

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Degradation of 3-nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzenetriol

Growth of Pseudomonas putida B2 in chemostat cultures on a mixture of 3-nitrophenol and glucose induced 3-nitrophenol and 1,2,4-benzenetriol-dependent oxygen uptake activities. Anaerobic incubations of cell suspensions with 3-nitrophenol resulted in complete conversion of the substrate to ammonia and 1,2,4-benzenetriol. This indicates that P. putida B2 degrades 3-nitrophenol via 1,2,4-benzenetriol, via a pathway involving a hydroxylaminolyase. Involvement of this pathway in nitroaromatic metabolism has previously only been found for degradation of 4-nitrobenzoate.Reduction of 3 nitrophenol by cell-free extracts was strictly NADPH-dependent. Attempts to purify the enzymes responsible for 3-nitrophenol metabolism were unsuccessful, because their activities were extremely unstable. 3-Nitrophenol reductase was therefore characterized in cell-free extracts. The enzyme had a sharp pH optimum at pH 7 and a temperature optimum at 25°C. At 30°C, reductase activity was completely destroyed within one hour, while at 0°C, the activity in cell-free extracts was over 100-fold more stable. The K_m values for NADPH and 3-nitrophenol were estimated at 0.17 mM and below 2 M, respectively. The substrate specificity of the reductase activity was very broad: all 17 nitroaromatics tested were reduced by cell-free extracts. However, neither intact cells nor cell-free extracts could convert a set of synthesized hydroxylaminoaromatic compounds to the corresponding catechols and ammonia. Apparently, the hydroxylaminolyase of P. putida B2 has a very narrow substrate specificity, indicating that this organism is not a suitable biocatalyst for the industrial production of catechols from nitroaromatics.     

Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoateto 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K_mand V_max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate.The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.     

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.     

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of β-Ketoadipate enol-lactone hydrolase and succinyl CoA: β-Ketoadipate CoA transferase

-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA:-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH₂-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by -ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.Dedicated to Roger Stanier with whom biochemistry was without tears (Stanier 1980)     

d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

The effect of a non-metabolizable analog on mandelate catabolism in Pseudomonas putida

Dl-2,3,4,5,6-pentafluoromandelic acid (PFM) specifically inhibits the growth of Pseudomonas putida (ATCC 12633) on medium containing mandelate as sole carbon and energy source by competitive inhibition of mandelate dehydrogenase. PFM is not metabolized and is neither an inducer of the mandelate catabolic enzymes nor an antagonist of induction. Mutants resistant to the inhibitory effects of PFM (PFM^r) were isolated; most prove to be superinducible, i.e. synthesize coordinately the mandelatespecific catabolic enzymes at elevated levels following induction. In at least one case the PFM^r mutation maps very near the structural genes that encode the enzymes functional in the first two steps of mandelate catabolism. It is reasoned that the PFM^r mutation is of the promotor type. Resistance to substrate analogs such as PFM offers a general method for isolation of regulatory mutants in catabolic metabolism.Dedicated to Prof. Roger Y. Stanier on the occasion of his 60th birthday     

Specificity of Lysine:N-Hydroxylase: A Hypothesis for a Reactive Substrate Intermediate in the Catalytic Mechanism

The recombinant cytoplasmic preparation of lysine:N⁶-hydroxlase catalyzes the conversion ofL-lysine to itsN⁶-hydroxy derivative when supplemented with the cofactors NADPH and FAD. A number of lysine analogs reflecting minor alterations in the inherent structural features of the amino acid as well compounds with relatively high affinity for lysine binding domains in other proteins were examined for their ability to serve as substrates of lysine:N⁶-hydroxylase. These studies have revealed that the enzyme does not tolerate any change in the structural features ofL-lysine, its preferred substrate, with the exception of the replacement of the C_γH₂-methylene group by sulfur, as in (S)-2-aminoethyl-L-cysteine.L-Norleucine is a potent inhibitor of the enzyme whileL-norvaline andL-α-aminobutyric acid do not exhibit such effect, indicating the importance of a C₄hydrophobic side chain for effective interaction with the enzyme. Among theN-alkyl amides of hydrophobic amino acids, onlyL-norleucine methylamide andL-α-aminobutyric acid ethylamide serve as moderate inhibitors of lysine:N⁶-hydroxylase. Based on the enzyme's stringent substrate specificity, a mechanism involving the conversion ofL-lysine to 2-aminocaprolactam prior to its oxygenation by the 4a-peroxyflavin intermediate in the catalytic cycle is proposed.     

Leaching of zinc from an industrial filter dust withPenicillium,Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

Summary Heterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.