Hydantoin-hydrolyzing enzyme

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Fig. 2. Reactions catalyzed by hydantoin-hydrolyzing enzymes (hydantoinases)...

Hydantoinases and decarbamoylases have been applied for the production of optically active amino acids from DL-5-monosubstituted hydantoins. A variety of enzymes have been reported elsewhere. Runser et al. [33] reported the occurrence of D-hydantoinase without dihydropyrimidinase activity. Watabe et al. [34] reported that an ATP-dependent hydantoin-hydrolyzing enzyme is involved in the L-amino acid production from DL-5-monosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme shows no stereospecificity. Hydan-toinase showing no stereospecificity and not requiring ATP was also reported [35]. Recently, hydantoin-racemizing enzymes were found [36,37], These enzymes make it possible to totally convert racemic substrates, which only slowly racemize under reaction conditions, to a single stereoisomer. The combinations of these hydantoin-transforming enzymes provide a variety of processes for optically active amino acid production (Fig. 4). 

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

Runser, S.M. Meyer, P.C., Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium sp. a hydantoinase with no... 

Hydantoinase process, outlined in Fig. 1, includes two hydrolases—hydantoin-hydrolyzing enzyme (hydantoinase) and AT-carbamoyl amino acid-hydrolyzing enzyme (carbamoylase)—and is one of the most efficient and versatile methods for the production of optically active a-amino acids. DL-5-Monosubstituted hydantoins, which are used as common precursors for the chemical synthesis of DL-a-amino acids [1], are the starting material of this enzymatic process. Keto-enol tautomerism is a typical feature of the hydantoin structure. Under neutral conditions, the keto form is dominant in alkaline solution, enolization between the 4 and 5 positions can occur, as has been concluded from the fact that optically pure hydantoins readily racemize. This feature is of practical relevance for the complete conversion of racemic hydantoin derivatives to optically pure L- or D-a-amino acids without any chemical racemization step. A variety of hydantoinase and carbamoylase with different stereospecificity were found. They are D-specific hydantoinase (D-hydantoinase), L-specific hydantoinase (L-hydantoinase), none-specific hydantoinase (DL-hydantoinase), D-specific carbamoylase (D-carbamoylase), and L-specific carbamoylase (L-carbamoylase). With the combination of these enzymes, optically pure amino acids are obtained from DL-5-monosubstituted hydantoins (Fig. 2). The wide substrate range of hydantoinases and carbamoylases also gives generality to the hydantoinase process. 

As described in this chapter, variety of hydantoin-hydrolyzing enzymes (Fig. 6) and N-carbamoyl amino acid amidohydrolases (Fig. 7) are involved in hydantoin transformation. The combinations of these enzymes provide a variety of prcxiesses for the production of optically pure a-amino acids (Fig. 8) [36,66-68]. The ability to completely convert a 100% hydantoin racemate into optically pure enantiomer renders these processes very attractive. Stereospecific D- or L-hydantoinase can produce optically pure iV-carbamoyl-D-or L-amino acid, respectively, from DL-5-monosubstituted hydantoin with 100% yield. The use of DL-hydantoinase together with stereospecific d- or L-caibamoylase also can provide optically pure d- or L-amino acid, respectively, from DL-5-monosubstituted hydantoin with 100% yield. Construction of recombinant microorganisms carrying these enzymes and immobilization of the cells or the enzymes could enhance the efficiency of the process as already demonstrated for D-/ -hydroxyphenylglycine production [69]. 

The hydantoinases hydrolyzing both d- and L-hydantoin were also found in several bacteria. They could be divided into two groups one needs ATP for its activity and the other does not. The ATP-requiring enzyme was purified and cloned from Pseudomonas sp. strain NS 671 [37,38]. The enzyme consists of two subunits with differing molecular mass of 76 and 65 kDa, and preferably hydrolyzes L-hydantoin. The enzyme that does not require ATP was purified and cloned from a moderate thermophilic bacterium B. stearothermophilus NS 1122A [39,40] and Arthrobacter sp. DSM 3745 [41]. The Bacillus enzyme is homotetrameric with molecular mass of 200 kDa. Although the monomer had no activity. 

Enzymatic Method. L-Amino acids can be produced by the enzymatic hydrolysis of chemically synthesized DL-amino acids or derivatives such as esters, hydantoins, carbamates, amides, and acylates (24). The enzyme which hydrolyzes the L-isomer specifically has been found in microbial sources. The resulting L-amino acid is isolated through routine chemical or physical processes. The D-isomer which remains unchanged is racemized chemically or enzymatically and the process is recycled. Conversely, enzymes which act specifically on D-isomers have been found. Thus various D-amino acids have been... 

The heterocyclic ring of hydantoins, like that of succinimides (see Sect. 4.4.2), is hydrolytically cleaved by dihydropyrimidine aminohydrolase (DHPase, EC 3.5.2.2). Since both hydantoins and succinimides are hydrolyzed by the same enzyme, it is not surprising that structural features, such as absolute configuration, ring-substitution, and TV-substitution, exhibit comparable influence on catalysis. 

It has been shown recently that papain exhibits hydantoinase activity. This enzyme of plant origin hydrolyzes not only 5-monosubstituted but also 5,5-disubstituted hydantoins to the corresponding N-carbamoylamino acids. Since chemical hydrolysis of the latter yields the corresponding amino acids, this approach may be of interest in amino acid synthesis [145],... 

The two groups of enzymes discussed here have attracted attention because both offer a useful broad spectrum of substrate specificity. They are grouped together because in the context of amino acid synthesis they form a natural pair. Amino acid hydantoins are convenient from the standpoint of organic synthesis. The hydantoinases cleave the ring, producing the A-carbamoyl derivative of the amino acid. This must then be further hydrolyzed to obtain the free amino acid, and this step is likely to be strictly enantioselective (Equation (10)). 

Decarbamoylation to D-amino acid was performed by treating the N-carbamoyl-D-amino acid with equimolar nitrite under acidic conditions [6]. But now, this step can also be carried out enzymatically. Recently, Shimizu and co-workers found a novel enzyme, D-decarbamoylase (IV-carbamoyl- n-amino acid amidohydrolase), which stereospecifically hydrolyzes JV-carbamoyl-D-amino acids, in several bacteria [7, 8], For example, Blastobacter sp. A17p-4 was found to produce D-decarbamoylase together with D-hydantoinase [8]. Therefore, a sequence of two enzyme-catalyzed reactions, the D-stereospecific hydrolysis of DL-5-(p-hydroxyphenyl) hydantoin and subsequent hydrolysis of the D-carbamoyl derivative to D-p-hydroxyphenylglycine, is possible (Fig. 1). Based on these results, a new commercial process for the production of D-p-hy-droxyphenylglycine has been developed [9]. 

One of the most widely used enzymatic methods for D-amino acid production is the hydantoinase process [4]. The great advantage of this process is that, potentially, any optically pure D-amino acid can be obtained using the corresponding substrate from a wide spectrum of D,L-5-monosubstituted hydantoins, which are readily accessible by chemical synthesis [5]. In this cascade of reachons the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme (D-hydantoinase). Further hydrolysis of the resulting N-carbamoyl D-amino acid to the free D-amino acid is catalyzed... 

System 1 was able to hydrolyze the 5-monosubstituted hydantoins faster than system 2 for the production of almost all the D-amino acids studied. System 1 was slightly slower than system 2 only for the production of the aromatic amino acids D-tyrosine and D-phenylglycine. This agrees with previously described results, finding that AtHyuAl enzyme (included in system 1) was more viable for industrial application than AtHyuA2 (included in system 2) due to its higher substrate affinity and racemization velocity [25]. 

Monosubshtuted hydantoins are a-amino acids cyclically protected at both the carboxyl- and the a-amino group. They can be easily prepared from an aldehyde and isocyanate or by the Bucherer-Bergs synthesis and similar methods. Indeed, the hydantoin synthesis is also a prachcal method for the preparahon of the racemic amino acid. Enzymes belonging to the dihydro-pyrimidinase family hydrolyze hydantoins to the carbamoyl amino acid. The latter can be hydrolyzed in turn to the amino acid by a second enzyme, a carbamoylase. Both enzymes can discriminate between enantiomers and, if their action is cooperative, either the L- or the D-amino acid can be obtained (Scheme 13.10) [36]. What makes the system of special interest is that the proton in the 5-position of the hydantoin ring (it will become the a-hydrogen in the a-amino acid) is considerably more acidic than conventional protons in amino acid esters or amides and much more acidic than the amino acid itself. Thus, the hydantoin can be often racemized in situ at slightly basic pH where the enzymes are stiU stable and active. If these condihons are met. 

D-Hydantoinases exist at different levels in many microorganism strains, and many of them have been cloned and overexpressed [103,122,123], This group of enzymes catalyzes enantioselective ring cleavage of 5-substituted hydantoins to give the corresponding D-A-carbamylamino acids that can be further hydrolyzed chemically or enzymatically to the free D-amino acids. The chemical... 

The reaction concept with this new hydantoinase-based biocatalyst is economically highly attractive since it represents a dynamic kinetic resolution process converting a racemic hydantoin (theoretically) quantitatively into the enantiomerically pure L-enantiomer [19]. The L-hydantoinase and subsequently the L-carbamoylase hydrolyze the L-hydantoin, l-11, enantioselectively forming the desired L-amino acid, l-2. In addition, the presence of a racemase guarantees a sufficient racemiza-tion of the remaining D-hydantoin, d-11. Thus, a quantitative one-pot conversion of a racemic hydantoin into the desired optically active a-amino acid is achieved. The basic principles of this biocatalytic process in which three enzymes (hydan-toinase, carbamoylase, and racemase) are integrated is shown schematically in Fig. 9. 

The six-membered ring systems 5,6-dihydropyrimidine, 5,6-dihydrouracil and 5,6-dihydrothymine can be hydrolyzed by the enzyme dihydropyrimidinase (E.C. 3.5.2.2), which is involved in the degradation of pyrimidine nucleotides. This widely spread, inducible catabolic enzyme is strictly D-selective in contrast to the L-selective dihydroorotase (E. C. 3.5.2.3), which is involved in the opposite anabolic pathway (see above). Another name often used in the literature for the dihydropyrimidinase is d-hydantoinase, because it is also able to hydrolyze D,L-5-monosubstituted hydantoin derivatives with high activity. Both reactions are shown in Fig. 12.4-7. 

Two other hydantoinases are described in the literature, which have not yet been listed in the Enzyme Nomenclature 9. Siedel et al. 41, Yamada et al. 42, 43 and Ogawa et al.[44 found a new ATP-dependent 1-methylhydantoinase with additional nucleoside-triphosphatase activity 45 in different bacteria. This inducible enzyme, which was also shown to act on unsubstituted hydantoin and 5-methylhydantoin 41, is involved in the degradation of creatinine after its deimination in the 2-position to 1-methylhydantoin, resulting in N-carbamoylsarcosine (N-carbamoyl-N-methylgly-cine) 42, 43 (see Fig. 12.4-9). It is associated with a so-called D-N-carbamoylsarcosine hydrolase 43, which eventually hydrolyzes N-carbamoylsarcosine to free sarcosine. Both enzymes can be used for monitoring creatinine levels in blood 41. ... 

Figure 12.4-11 gives a survey of the substrates accepted by the different dihy-dropyrimidinase or D-hydantoinase preparations The differences between the en-zyme preparations from mammalian and microbial sources are discussed in more detail in reference13, but D-hydantoinases or dihydropyrimidinases, respectively, seem to have the following in common (i) a wide substrate specificity, (ii) metal dependence and (iii) that they are strictly D-specific. Preferably, cyclic amides are hydrolyzed at pH values around 8.5. Furthermore, most of the enzymes are also described to be able to catalyze the hydantoin formation the optimal pH of this reaction is neutral or weakly acidic. 

Substituted hydantoin derivatives have been used as precursors for d- and L-amino acids in chemical synthesis. However, they are hydrolyzed enantioselectively by the enzymes named hydantoinases some act specifically on D-5-substituted hydantoins, and others on the L-isomers. N-Carbamoyl amino acids formed are also hydrolyzed enantiospecifically by N-carbamoyl amino acid amidohydrolases to produce d- or l-amino acids (Fig. 17-15). Since the Kanegafuchi Chemical Industry, Japan, commercialized an enzymatic procedure for the production of D-p-hydroxyphenylglycine, which is a building block for the semisynthetic P-lactam antibiotic amoxycillin, various processes for amino acid production by means of hydantoinases have been developed 131 133. ... 

A process for the production of D-a-amino acids has been developed by Roche Diagnostics based on the enzyme D-hydantoinase [115]. The recombinant protein was covalently fixed onto a carrier and used for the synthesis of a broad range of natural and artificial D-amino acids (132, Scheme 41). Starting from racemic hydantoins d/l-130, the enzyme exclusively hydrolyzed d-130 to 131, and new d-130 was internally produced by continuous in situ racemization of l-130. The process worked especially well with 5-(p-hydroxyphenyl)- and 6-phenylhydantoin, affording the corresponding amino acids 132 in high yield and optical purity. The number of reuse cycles until 50% of the initial enzyme activity was reached was calculated to be as high as - 200. Unfortunately, this process has never been used in the production of D-amino acids, as diazotation was found to be too noxious and complicated. 

The enantiomerically pure amino acids also can be produced through a similar synthetic pathway catalyzed by enzymes. For example, reaction of hydrogen cyanide with benzaldehyde catalyzed by either (R)- or (S)-oxynitrilase enzyme yields the enantiomeric cyanohydrins (R)- or (S)-mandelonitrile. Alternatively, by adding carbon dioxide and hydrogen cyanide and ammonia as feedstocks, aldehydes can be converted to hydantoins (4-alkylimidazolidine-2,5-diones), which can be then hydrolyzed with either D- or L- hydan-toinases to produce D- or L-a-amino acids, respectively. 

Recently, the novel enzyme D-carbamoylase, which stereospecifically hydrolyzes iV-car-bamoyl-D-amino acids, was found in several bacteria [13,14]. Therefore, a sequence of two enzyme-catalyzed reactions, the D-stereospecific hydrolysis of DL-5-(p-hydroxy-phenyl)hydantoin and subsequent hydrolysis of the D-carbamoyl derivative to D-p-hydrox-... 

Takahashi et al. [6] revealed that in Pseudomonas putida (= P. striata) BFO 12996 d-hydantoinase is identical with dihydropyrirnidinase (EC 3.5.2.2), which catalyzes the cyclic ureide-hydrolyzing step of the reductive degradation of pyrimidine bases (Fig. 4). The same results were obtained for other hydantoinases from Pseudomonas sp. [22,23], Com-amonas sp. [23], Bacillus sp. [9], Arthrobacter sp. [24], Agrobacterium sp. [22], and rat liver [25]. From these results, it is proposed that D-amino acid production from dl-5-monosubstituted hydantoins involves the action of the series of enzymes involved in the pyrimidine degradation pathway [24,26,27], However, this contenticm has remained moot because of a lack of systematic studies on the enzymes involved in these transformations [28]. 

A novel enzyme imidase was found in Blastobacter sp. A17p-4 [42]. The imidase hydrolyzes dihydrouracil, hydantoin, and various cyclic imides very effectively (Fig. 5a). The imidase catalyzes the first reaction of the microbial cyclic imide degradation [43]. 

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