Hydantoinase reactions catalyzed

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

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Racemic hydantoins result from the reaction of carbonyl compounds with potassium cyanide and ammonium carbonate or the reaction of the corresponding cyanohydrins with ammonium carbonate (Bucherer-Bergs reaction). Hydantoins racemize readily under basic conditions or in the presence of hydantoin racemase, thus allowing DKR (Figure 6.43). Hydantoinases (EC 3.5.2.2), either isolated enzymes or whole microorganisms, catalyze the hydrolysis of five-substituted... 

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]. 

Figure 12.4-7. Analogy between dihydropyrimidinase- and D-hydantoinase-catalyzed reactions.

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. 

Figure 12.4-17. Proposed reaction mechanism catalyzed by the hydantoinase after binding of the substrate, an electrophilic residue (or zinc) stabilizes the negative charge of the carbonyl oxygen. Zinc-bound water is activated and performs a nucleophilic attack on the C4 carbon...

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