In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.

L-Hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity and crystallized from polyethylene glycol solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the sitting-drop variant of the vapour-diffusion method. X-ray diffraction studies show that the crystals belong to the monoclinic space group P2(1) with a = 111.2, b = 74.4, c = 146.5 A and beta = 106.7 degrees. Its asymmetric unit contains four monomers related by 222 symmetry. The crystals diffract to at least 2.6 A.

Arthrobacter aurescens strain TC1 metabolizes atrazine to cyanuric acid via TrzN, AtzB, and AtzC. The complete sequence of a 160-kb bacterial artificial chromosome clone indicated that trzN, atzB, and atzC are linked on the A. aurescens genome. TrzN, AtzB, and AtzC were shown to be functional in Escherichia coli. Hybridization studies localized trzN, atzB, and atzC to a 380-kb plasmid in A. aurescens strain TC1.

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 degrees C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg2+, Mn2+, and Ca2+ while strongly inhibited by 5 mM Cu2+ and SDS.

Previously, a di-D-fructofuranose 1,2':2,3' dianhydride (DFA III)-producing strain, Arthrobacter aurescens SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in Escherichia coli. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55�XC, with specific activity of 232 U mg-1. Km and Vmax for DFA III were 30.7 �� 4.3 mM and 1.2 �� 0.1 mM min-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from Bacillus sp. snu-7 as a template. It was suggested that A. aurescens DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from Bacillus sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of A. aurescens DFA IIIase.

Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

Difructose anhydride (DFA) III is a natural and low-calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over-expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL(-1) . This value was 4.1-fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 �XC, and retained 81.3% of its initial activity after incubation at 60 �XC for 4 h. IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over-expressed in E. coli. IFTase was reported for the first time to be over-expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production.

The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively. A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5). Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution. We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria. Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.

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.