Oliver May

Microbial hydantoinases--industrial enzymes from the origin of life?

Abstract Hydantoinases are valuable enzymes for the production of optically pure d- and l-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5′-monosubstituted hydantoins and are therefore classified in the EC nomenclature as cyclic amidases (EC 3.5.2.). In the EC nomenclature, four different hydantoin-cleaving enzymes are described: dihydropyrimidinase (3.5.2.2), allantoinase (EC 3.5.2.5), carboxymethylhydantoinase (EC 3.5.2.4), and N-methylhydantoinase (EC 3.5.2.14). Beside these, other hydantoinases 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 classified. An important question is whether the distinct hydantoinases, which are frequently classified as l-, d-, and non-selective hydantoinases depending on their substrate specificity 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 sequence 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 divergently from a common ancestor and (iii) that they share a metal-binding motif consisting of conserved histidine residues. The difference in enantioselectivity used for the classification of hydantoinases on the basis of their biotechnological value does not reflect 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.

Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745 : Purification and characterization as new member of cyclic amidases

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Catalytic and structural function of zinc for the hydantoinase from Arthrobacter aurescens DSM 3745

Abstract Metal dependency of the hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic studies of metal/chelator-caused enzyme inactivation, denaturation and reactivation, accompanied by the identification of specific metal binding ligands. The enzyme can be inactivated by metal chelating agents and—apart from the loss of its activity—completely dissociates into its subunits. Enzyme activity can be restored from recollected monomers by the addition of cobalt, manganese or zinc-ions, whereas nickel and magnesia remain ineffective. Subjection of the hydantoinase to metal analysis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an essential role not only for the catalytic activity but also for the stabilization of the active quarternary structure of the hydantoinase. Histidine-specific chemical modification of the enzyme causes a complete loss of the catalytic activity and reveals histidine residues as putative zinc binding ligands. Both, the metal/chelator-caused enzyme inactivation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that at least one metal-ion acts specifically near or at the active site of the enzyme.

The hydantoin amidohydrolase from Arthrobacter aurescens DSM 3745 is a zinc metalloenzyme

Abstract The hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 was purified to homogeneity and subjected to metal analysis under atomic absorption spectrometry (AAS) and inductive coupled plasma–atomic emission spectrometry (ICP–AES). Three independent preparations of homogeneous enzyme indicated that 1 mol of the active enzyme contains 10 mol zinc ions. This corresponds to 2.5 mol zinc per mol subunit, since the hydantoinase consists of four identical subunits. Only trace amounts of manganese, magnesia, nickel and cobalt were detected. Other metals were either absent or existed below detection levels.

A new method for the detection of hydantoinases with respect to their enantioselectivity on acrylamide gels based on enzyme activity stain

A new and highly sensitive method was developed for the identification of hydantoinases on acrylamide gels. For this purpose, cell-lysates from different natural isolates are subjected on PAGE under non-denaturating conditions. The respective localisation of the hydantoinase is obtained by in situ product precipitation during the reverse enzyme reaction: in contrast to the used substrate (N-carbamoyltryptophan), the product (indolylmethylhydantoin) is barely soluble and gives a dense precipitation dot caused by crystallisation of the product inside of the polyacrylamide gel at the position corresponding to the location of the enzyme. This method can also be used for the subsequent differentiation between L- and D-selective hydantoinases, since L- or D-carbamoyltryptophan is used as substrate.