Hydantoin-transforming enzymes

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). 

Figure 8 Optically pure a-amino acid production processes by combinations of hydantoin-transforming enzymes.

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]. 

As we presented here, potential of stereoselective synthesis using hydantoinases and carbamoyl es is based on the unusual diversity of the microbial enzymes and their strict sj ificity. Application of microbial hydantoin transformation is a good example of the appficabihty of enzymes from a microbially diverse world. 

The use of a whole cell allows for a required enzyme cofactor to be regenerated. In other cases, it allows for several enzymes to work in parallel and to perform many complex transformations. An example is provided by the synthesis of D-amino acids from hydantoins (Fig. 3). The carbomylase drives the reaction to completion as carbon dioxide and ammonia are evolved. The same approach has been used with the l-versions of the enzymes to synthesize L-amino acids (14, 42, 55). 

Recombinant whole cells in particular turned out to be very attractive for bio-transformations in which more than one recombinant enzyme is needed such as redox reactions with in situ cofactor regeneration or hydrolysis with mrdtiple enzymes. With respect to the latter one, the dynamic kinetic resolution of hy-dantoins by a whole-cell catalyst that simultaneously overexpresses a racemase, a hydantoinase and a carbamoylase is a popular and industrially relevant example (Scheme 2.8) [23,24]. These cells convert a racemic hydantoin (an easily accessible substrate) to the corresponding enantiomerically pure d- or L-amino add with both high conversion and enantioselectivity. 

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]. 

An ATP-dependent amidohydrolase A-methylhydantoin amidohydrolase, which catalyzes the second-step reaction in the degradation route from creatinine to glycine, via N-methylhydantoin, iV-carbamoylsarcosine, and sarcosine as successive intermediates [44-52] (Fig. 5b), was found in Pseudomonas putida 77 [44,45]. The enzyme is inducible only with the presence of creatinine and iV-methylhydantoin, suggesting that the role of this enzyme is in the transformation of creatinine [53]. The ATP-dependent hydrolysis of 5-monosubstituted hydantoins, e.g., 5-methylhydantoin, by the enzyme proved to be L-iso-mer-specific [53] ... 

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