5-monosubstituted hydantoin substrates

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]. 

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). 

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... 

Additional binding experiments conducted by fluorescence measurements with C76A mutant and D- and L-isopropylhydantoin and L-ethylhydantoin (D-ethylhydantoin is an inhibitor) showed that this mutant is unable to bind the D-isomers of the substrates. The same experiments carried out with C181A mutant proved that this mutant was not able to bind the L-isomers. These results indicate that cysteine 76 is responsible for the recognition of D-isomers of the 5-monosubstituted hydantoins, whereas cysteine 181 recognizes the L-isomers. 

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]. 

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. 

D-Hydantoinase was isolated from mammalian cells as well as from bacteria. The bovine liver enzyme is identical to dihydrop3nimidmase and consists of four subunits [31]. It was reported to have four Zn which are tightly bound sirnilar to that of rat dihydropyiimi-dinase [32]. Bacterial D-hydantoinase was isolated from various sources such as P. putida (= P. striata) IFO 12996 [6], Pseudomonas sp. AJ 11220 [27], Pseudomonas fluorescens DSM 84 [33], Bacillus stearothermophilus SDl [34], Blastobacter sp. A17p-4 [35], and Arthrobacter crystallopoietes AM2 [24]. The substrate specificities of some D-hydanto-inases are summarized in Table 3. It can be seen that all bacterial D-hydantoinases other than the enzyme from Agrobacterium sp. IP 1-671 [30] are rather similar to file mammalian dihydropyrimidinase. D-Hydantoinase from Agrobacterium sp. IP 1-671 is specific for D-5-monosubstituted hydantoin and has no dihydropyrimidinase activity. All bacterial d-hydantoinases other than the thermostable enzyme from B. stearothermophilus are homo-tetramer with molecular masses of 190-260 kDa. The enzyme from B. stearothermophilus is homodimer with a molecular mass of 126 kDa. These enzymes activities require divalent cations such as Mg, Mn, Fe, Co, or Zn for their expression of maximum activity. 

Allantoinase (EC 3.5.2.5) is widely distributed in nature and plays an important role in the degradation of purine nucleosides (Fig. 5d). Investigation on substrate specificity of allantoinase is limited. However, it would be of interest to test allantoinase concerning the hydrolysis of various DL-5-monosubstituted hydantoins. 

In optically pure a-amino acid production from DL-5-monosubstituted hydantoins, the wide applicability to a broad substrate range is valuable especially for the production of D-a-amino acids [70] and unnatural L-a-amino acids (Fig. 9), e.g., D-p-hydroxyphen-ylglycine [71], D-phenylglycine [71], substituted L-phenyManine such as L-p-chlorophen-ylalanine [72] and p-trimethylsilylphenylalanine [73,74], L-a- and p-naphthylalanine [75], an A -methyl-D-aspartate receptor antagonist, (2R, 4R, 55)-2-amino-4,5-(l,2-cyclohexyl)-7-... 

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