MINI-REVIEW C. Syldatk á O. May á J. Altenbuchner á R. Mattes M. Siemann Microbial hydantoinases ± industrial enzymes from the origin of life? Received: 24 August 1998 / Received revision: 9 November 1998 / Accepted: 21 November 1998 Abstract Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hy- dantoin or 5¢-monosubstituted hydantoins and are therefore classi®ed in the EC nomenclature as cyclic amidases (EC 3.5.2.). In the EC nomenclature, four dierent hydantoin-cleaving enzymes are described: di- hydropyrimidinase (3.5.2.2), allantoinase (EC 3.5.2.5), carboxymethylhydantoinase (EC 3.5.2.4), and N-met- hylhydantoinase (EC 3.5.2.14). Beside these, other hy- dantoinases with known metabolic functions, such as imidase and carboxyethylhydantoinase and enzymes with unknown metabolic function, are described in the literature and have not yet been classi®ed. An important question is whether the distinct hydantoinases, which are frequently classi®ed as L-, D-, and non-selective hy- dantoinases depending on their substrate speci®city and stereoselectivity, are related to each other. In order to investigate the evolutionary relationship, amino acid sequence data can be used for a phylogenetic analysis. Although most of these enzymes only share limited se- quence homology (identity<15%) and therefore are only distantly related, it can be shown (i) that most of them are members of a broad set of amidases with similarities to ureases and build a protein superfamily, whereas ATP-dependent hydantoinases are not related, (ii) that the urease-related amidases have evolved di- vergently from a common ancestor and (iii) that they share a metal-binding motif consisting of conserved histidine residues. The dierence in enantioselectivity used for the classi®cation of hydantoinases on the basis of their biotechnological value does not re¯ect their evolutionary relationship, which is to a more diverse group of enzymes than was assumed earlier. This protein superfamily probably has its origin in the prebiotic conditions of the primitive earth. Introduction According to the EC nomenclature, hydantoinase is an alternative name for dihydropyrimidinase (EC 3.5.2.2). This cyclic amidase catalyses the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and also uses dihydrothymine and hydantoin (also called imidazoli- dine-2,4-dione or 2,4-diketotetrahydro-imidazole; Ware 1950) as alternative substrates (Webb 1992). The cata- lysed reactions are shown in Fig. 1. Not only their important metabolic function in the reductive pathway of pyrimidine degradation (see 1.2), but also their biotechnological application in the pro- duction of optically pure amino acids has generated considerable interest in these enzymes (Syldatk et al. 1992a, b; Syldatk and Pietzsch 1995; Shimizu et al. 1997). This biotechnological interest has led to the iso- lation of many dierent enzymes all hydrolysing hydantoin and/or 5¢-monosubstituted hydantoin deriv- atives. It is common for the literature to classify these enzymes according to their stereoselectivity as D-, L-, and non-selective ``hydantoinases''. The name hy- dantoinases, frequently used for this group of enzymes, tends to be misleading because it could imply that hy- dantoinases are homologous or similar and only dier in their stereoselectivity. One might even believe that these ``hydantoinases'' are identical to dihydropyrimidinase which also has the alternative name of hydantoinase in EC nomenclature. Therefore it should be pointed out that, in this review, the name hydantoinase will be used for all enzymes that hydrolyse hydantoin and/or Appl Microbiol Biotechnol (1999) 51: 293±309 Ó Springer-Verlag 1999 C. Syldatk (&) á O. May 1 á M. Siemann Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany e-mail: syldatk@ibvt.uni-stuttgart.de Tel.: ++49-711-685-4574 Fax: ++49-711-685-5164 J. Altenbuchner á R. Mattes Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Present address: 1 Division of Chemistry and Chemical Engineering, Caltech 210-41, Pasadena, CA 91125, USA