ABSTRACT
Hydantoinases and dihydropyrimidinases belong to the amidohydrolase superfamily and are acting on carbon-nitrogen bonds of cyclic amides (Holm and Sander, 1997). To date, several crystal structures of dihydropyrimidinases and hydantoinases are solved and listed in the Protein Data Bank (www.PDB.org, (Berman et al., 2000)). The subunits of these enzymes consist of two domains: a β-sandwich domain and a distorted (α/β)8 barrel domain harboring the catalytic core. This active site contains a binuclear metal center and thus explains the dependence on divalent metal ions of hydantoinases and dihydropyrimidinases. Except for the D-hydantoinase of Bacillus sp. AR9, all described enzyme crystal structures accommodate a posttranslational carboxylated lysine residue in the active site (Kishan et al., 2005; Seibert and Raushel, 2005). Another feature is the highly conserved GXXDXHXH-sequence motif that is involved in the coordination of the metal ions in the active site (Holm and Sander, 1997; May et al., 1998a). The substrate recognition and enantioselectivity of hydantoinases/dihydropyrimidinases is assumed to be determined by three so-called stereochemistry gate loops (SGLs). These SGLs are structurally conserved among hydantoinases/dihydropyrimidinases and form a hydrophobic pocket suggested to recognize the side chain of 5-monosubstituted hydantoins. The specific amino acid composition of these SGLs defines the unique substrate specificity of each enzyme (Cheon et al., 2002; Cheon et al., 2003; Cheon et al., 2004; Lo et al., 2009). The C-terminus of hydantoinases/dihydropyrimidinases is reported to be nonessential for catalysis and to be nonconserved among the known eukaryotic and prokaryotic enzymes (Kim and Kim, 1998). However, Martinez-
Rodriguez et al. (2010b) found that the C-terminal regions of hydantoinases/dihydropyrimidinases of α-Proteobacteria are highly conserved. The C-termini are assumed to influence the oligomeric structure of the enzyme (Kim and Kim, 1998). Most hydantoinases and dihydropyrimidinases are homotetramers or homodimers (Schnackerz and Dobritzsch 2008; Syldatk et al., 1999). It was shown by Yoon et al. (2003) that a mutation of several C-terminal residues of the hydantoinase from Bacillus stearothermophilus SD1 led to a change of the oligomeric structure of the enzyme from a homotetramer to a homodimer. 22.3.4 Classification
Hydantoinases are commonly subdivided into D-, L-, or non-selec-tive enzymes (LaPointe et al., 1994). This system is not reliable because several hydantoinases were shown to have a substratedependent enantioselectivity. Yokozeki and Kubota (1987) report-ed for the enzyme of Flavobacterium sp. AJ-3912 an L-selective cleavage of indolylmethylhydantoin but a D-selective conversion of benzyloxymethylhydantoin. The hydantoinase of Arthrobacter aurescens DSM3745 exhibited no selectivity for methylthioethyl-hydantoin but showed an L-selectivity for indolylmethylhydan-toin (May et al., 1998b). For the enzyme of Brevibacillus agri NCHU1002 a D-specific conversion of methylthioethylhydantoin and p-hydroxyphenylhydantoin was demonstrated in contrast to a non-selective cleavage of 5-phenylethylhydantoin (Kao et al., 2008).
