Genes responsible for hydantoin degradation of a halophilic Ochrobactrum sp. G21 and Delftia sp. I24 — New insight into relation of d-hydantoinases and dihydropyrimidinases

Delftia sp. I24 and a moderately halophilic Ochrobactrum sp. G21 are able to hydrolyse dihydropyrimidines and hydantoins D-specific. The genes being with the utmost probability involved in dihydropyrimidine and hydantoin degradation of these two microorganisms were cloned into an appropriate vector and transformed into E. coli. The putative gene cluster of Delftia sp. I24 included four genes: an incomplete NADPH-dependent glutamate synthase (gltB), dihydropyrimidine dehydrogenase (pydA), permease (hyuP) and an incomplete d-hydantoinase (hyuH). The hydantoinase gene sequence was completed by PCR amplification. The putative gene cluster of Ochrobactrum sp. G21 comprised nine ORFs, six being potentially involved in hydantoin-hydrolysation: carbamoylase (hyuC), d-hydantoinase (hyuH), two transporters (OrfS1 and OrfS2) and two permeases (hyuP1 and hyuP2). Expression of the d-hydantoinases from Delftia sp. I24 and from Ochrobactrum sp. G21 in E. coli, followed by biotransformation assays confirmed hydantoinase activity. This is the first report of the genetical organization of hydantoin-degradation within the genera Delftia and Ochrobactrum. Phylogenetic analysis of the two “novel” hydantoinases and known hydantoinases and dihydropyrimidinases, including putative protein sequences, revealed that they can be classed with some exceptions in the following groups: l-hydantoinases (l-Hyd), Rhizobiales family (Rhizo-Fam), Comamonadacae family (Com-Fam), Pseudomonas family (Pseud-Fam), Bacilli family (Bac-Fam) and Agrobacterium family (Agro-Fam). The highly conserved “histidine motif” for the superfamily of amidohydrolases could be found for all hydantoinases of this study but differences were found in the substrate recognition sites, whereas some of the above mentioned groups showed to posses the same recognition sites as known hydantoinases.