3 AMINOHYDROLASES ACTING ON ADENINE, ADENOSINE AND THEIR DERIVATIVES

BACKGROUND
Adenine and adenosine-acting aminohydrolases are important groups of enzymes responsible for the metabolic salvage of purine compounds. Several subclasses of these enzymes have been described and given current knowledge of the full genome sequences of many organisms, it is possible to identify genes encoding these enzymes and group them according to their primary structure.


METHODS AND RESULTS
This article is a short overview of the enzymes classified as adenine and adenosine deaminase. It summarises knowledge of their occurrence, genetic basis and their catalytic and structural properties.


CONCLUSIONS
These enzymes are constitutive components of purine metabolism and their impairment may cause serious medical disorders. In humans, adenosine deaminase deficiency is linked to severe combined immunodeficiency and as such the enzyme has been approved for the first gene therapy trial. The role of these enzymes in plants is unclear, since the activity was has not been detected in extracts and putative genes have not been yet cloned and analyzed. A literature search and amino acid identity comparison show that Ascomycetes contain only adenine deaminase, but not adenosine deaminase, despite the fact that corresponding genes are annotated in databases as the adenosine cleaving enzymes because they share the same conserved domain.


INTRODUCTION
Out of 44 enzymes systematically labelled as aminohydrolases in the Enzyme Commission, 25 are classifi ed as deaminases.These enzymes control important pathways in the metabolism of the components of nucleic acids 1 , Fig. 1A.Genetic defi ciency in these pathways in humans results in serious diseases, such as severe combined immunodefi ciency caused by diminished activity of adenosine deaminase.Six of the known deaminases are involved in the salvage of adenine and its derivatives and nitrogen catabolism.This review focuses on the enzymes adenine deaminase and adenosine deaminase and covers recent progress in understanding their genetic origin and biochemical properties.

ADENINE DEAMINASE
Adenine deaminase (alternatively also adenine aminohydrolase or adenase, ADE, EC 3.5.4.2) catalyzes the hydrolytic cleavage of adenine to produce hypoxanthine and ammonia (Fig. 1B).ADE genes have been found in prokaryotes and lower eukaryotes such as yeasts or protozoa.Until recently, genes coding eukaryotic adenine deaminase were unknown, but genetic and physiological evidence indicated that these enzymes are present in Ascomycetes.Surprisingly, when some putative fungal adenosine deaminases (ADA, EC 3.5.4.4) have been cloned and expressed in E. coli, they exhibited adenine deaminase activity 2 .At the amino acid sequence level, bacterial adenine deaminases are more closely related to allantoinases, ureases and dihydroorotases than to fungal enzymes 2 .Of this enzyme class, a structure of bacterial ADE from Enterococcus faecalis has been the fi rst and so far the only one to become available 3 (in October 2006), Fig. 2A.

Substrates, activating compounds and inhibitors of adenine deaminase
Adenine deaminases have been detected in diff erent microorganisms, such as Pseudomonas synxantha 4 , Bacillus subtilis 5 , Leishmania donovani 6 , E. coli 7 and Azotobacter vinelandii 8 .Some of the enzymes were partially purifi ed and characterized in terms of specifi c activity and K m value.The enzymatic activity showed a maximum between pH 6 (C.fasciculata 9 ) and pH 9 (P.synxantha 10 ).Optimal temperature was found to be diverse, i.e. 40-45 °C for ADE from P. synxantha, but 60 °C (ref. 7) for the E. coli enzyme.Genes coding for ADE from E. coli 7 , S. cerevisiae 11 and B. subtilis 5,12 have been cloned and overexpressed in modifi ed E. coli.

Genetic relationship of adenine deaminases
Protein BLAST results of prokaryotic and eukaryotic ADEs clearly show that fungal enzymes are only weakly similar to bacterial and archaeal ones (Tab.1).Diff erences between archaeal and bacterial ADEs themselves are moderate, suggesting that they have close genetic origin as shown by phylogenetic analysis (Fig. 3A).These prokaryotic proteins form a separate group that is clearly distinct from any other ADE or ADA enzymes as indicated by low degree of amino acid sequence identity.Known eukaryotic ADE enzymes share a very high degree of similarity with putative fungal ADAs and they also contain conserved amino acid domains that associate them with bacterial and mammalian ADAs as well.

Protein structure of adenine deaminase
The enzyme AAH1 from Saccharomyces cerevisiae is a typical representative of ADEs from lower eukaryotes.The gene coding for this protein (GeneID: 855581) is located on chromosome XIV (NC_001146) at the position form 359598 to 360641 (1043 bp) and has no introns.According to amino acid domain search, the enzyme belongs to the metal-dependent hydrolase superfamily, adenosine deaminase (ADA) family and more specifi cally to the sub-family of α/β barrel enzymes, adenine deaminase family 2. The main structural feature of this protein is the conserved domain cd01320 that is characteristic for a monomeric zinc-dependent protein, which catalyzes the irreversible hydrolytic deamination of both adenosine, as well as 2′-deoxyadenosine, to ammonia and inosine or deoxyinosine, respectively.The same domain is present in the majority of adenosine deaminases.
A typical representative of bacterial ADEs is a yicP cryptic adenine deaminase from E. coli K12.The encoding gene (GeneID: 945851) is localized on the genome at the position from 3841987 to 3843753 (1766 bp).The enzyme belongs to the same superfamily, adenine deaminase family 1 2 and contains the conserved domain cd01295 (AdeC: adenine deaminase directly deaminating adenine to form hypoxanthine), which is diff erent from the cd01320 domain of AAH1.Similar domain architecture is found for the enzyme from Enterococcus faecalis, the structure of which has been solved recently (Fig. 2A).

ADENOSINE DEAMINASE
Adenosine deaminase (alternatively also adenosine aminohydrolase, ADA, EC 3.5.4.4) catalyzes irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and deoxyinosine.This kind of enzyme is present in prokaryotic and eukaryotic organisms in diff erent forms and belongs to the family of α/β barrel enzymes 2 similarly to the adenine deaminases.Given their widespread occurrence and importance in the medical fi eld, these enzymes have been investigated more profoundly than adenine deaminases.ADA is found in a variety of prokaryotes and eukaryotes (insects, lower vertebrates, fi sh, mammals and possibly also in plants).
In prokaryotes and eukaryotes, proteins that exhibit adenosine deaminase activity include not only the classical ADA (EC 3.5.4.4), but also ADA regulatory proteins (only in prokaryotes) with diff erent conserved domains (EC 2.1.1.63)and tRNA-specifi c adenosine deaminases (ADATs), which are structurally very similar to ADAs.ADAT was the fi rst prokaryotic RNA editing enzyme to be identifi ed in E. coli 15 .As described above, fungal ADEs are very similar at the amino acid sequence level to microbial ADAs and for this reason if classifi ed solely on sequence homology could be easily mistaken.

Mammalian adenosine deaminase
Two different isoenzymes 16 of ADA, ADA1 and ADA2, found in higher eukaryotes are encoded by diff erent genes 17 .In humans, almost all ADA activity is attributed to a single-chain Zn-binding protein ADA1, whereas ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages 18 .ADA1 is expressed in all human tissues, its activity levels being relatively high in thymus and duodenum (about 10 nkat/mg), whereas more than 500-fold lower activity was reported in liver 19 .The molecular basis for tissue-specifi c expression of ADA has been studied by analyzing ADA1 gene promoter and interactions of nuclear proteins with enhancer elements 20 .Specifi c regulatory regions of the gene coding for ADA1 in humans were investigated in transgenic experiments using ADA1 gene and promoter fragments linked to a bacterial chloramphenicol acetyl transferase (CAT) reporter gene 20 .A 232-bp region (-211 to +11) from the ADA1 gene promoter confers high basal reporter gene (CAT) expression in transient transfection assays in mammalian cells 21,22 .
Mutations in the ADA1 gene that block its expression cause severe combined immunodefi ciency, whereas mutations leading to overexpression cause hemolytic anemia.Lack of ADA1 activity leads to an accumulation of dATP that causes inhibition of the ribonucleotide diphosphate reductase activity, the enzyme that synthesizes DNA and RNA which are required in a large amount during lymphocyte proliferation.There is also some evidence that the other diff erent allelle (ADA2) may lead to autism.
Recently, ADA2 has been identifi ed as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms 18 .
Two additional types of adenosine deaminase are found in higher eukaryotes, ADATs and ADARs.Adenosine deaminase acting on RNA (ADAR) has the ability to deaminate adenosines in any long double-stranded RNA and converting them to inosines.ADARs are commonly found in animals, but not known in other organisms.ADARs are presumed to evolve from the adenosine deaminases acting on tRNAs (ADATs), by steps including fusion of two or more double-stranded-RNA binding domains to a common type of zinc-containing adenosinedeaminase domain 23 .
Metals ions in specifi c concentrations are in some cases essential for the activity.MgCl 2 and CoSO 4 had a remarkable activating eff ect on ADA from Aspergillus terricola 36 .Fe 3+ or Sn 2+ are promoting the enzymatic reaction of Nocardioides sp.J-326TK 31 and Streptomyces sp. 32adenosine deaminases.For Bacillus cereus enzyme, the activity is stabilized by NH 4 + or K + , while it is irreversibly lost in the absence of these or a few other monovalent cations 37 .Activating compounds were also described for the enzymes from Argopecten irradians concentricus (NH 4 Cl, (NH 4 ) 2 SO 4 ) 38 , Candida albicans enzyme (8-aza-adenine, adenine, AMP, ATP, IMP, inosine, N-acetyl-D-glucosamine) 39 and Mus musculus (dibutyryl-cAMP) 40 .
The value of pH optimum signifi cantly varies among enzymes from diff erent sources, but usually confi nes to the range between pH 5 and 8.The pH range to observe enzymatic activity was shown to be pH 5-9 for Bos taurus ADA 41 , pH 4-8 for Candida albicans 39 and pH 3.5-5.5 for Streptomyces sp. 32enzyme.Temperature optimum ranges from 25 °C for ADA from camel (Camelus dromedaries) 42 to 55 °C for the one from Streptomyces sp. 32.
As a part of a program to explore structure-activity relationships for extremely tight binding of coformycin inhibitors to adenosine deaminase, a series of analogues containing the imidazo[4,5-e][1,2,4]triazepine ring system was synthesized and showed to display inhibitory properties towards mammalian ADA in in vitro tests 48 .

Genetic relationship and structures of adenosine deaminases
Human ADA1 gene is located on the chromosome 20 (20q12-q13.11).The gene is 32,213 bp long and contains 11 introns, the coding sequences being only 4,53 % of the whole gene.The encoded protein belongs to the metallodependent hydrolase superfamily, adenosine deaminase (ADA) family and contains adenosine deaminase (ADA) conserved domain cd01320.
Typical prokaryotic gene representative, ADA gene from Escherichia coli K12, is located in the position from 1700257 to 1701258 of the genome (1001 bp).The encoded enzyme contains the same conserved domain as a human ADA.
Protein structures have been solved for prokaryotic and eukaryotic ADAs, a variety of domains and chains of ADAR, and for four chains of bacterial ADAT.No structure of a plant enzyme of this class is known.Comparison of protein structures of three typical representatives of these enzymes is shown in Fig. 2B.Sequences of adenosine deaminase coding genes of prokaryotes and eukaryotes are more related each other than those of adenine deaminases.The sequences of the genes from higher eukaryotes, mammalians particularly, are almost identical.At protein level, the degree of identity among mammalian enzymes is very high and clearly separates this group from the others (Tab.2).The same relationship is shown in the phylogenetic tree (Fig. 3B).Noticeably, fungal enzymes form group, which is the least similar with the others.Although these enzymes are annotated as ADAs, there is actually no experimental evidence that the respective proteins show ADA-like activity.Contradictory to the annotation, the enzyme from Schizosaccharomyces pombe (SPBC1198.02)shows no activity with adenosine, but readily acts on adenine 2,54 , thus being ADE not ADA.It is likely that also other closely related enzymes from Ascomycetes possess ADE, but not ADA activity and maybe adenosine deaminases are not presented in lower eukaryotes at all.These genes from lower eukaryotes contain no introns, but introns occupy about 95 % of the DNA sequence in mammalian ADA genes.Typically, the ADA gene has 11 introns.Dog ADA gene contains an additional 40 bp intron that may have evolved by an excision from the 5'-part of the 9 th intron.Mammalian ADA genes range from 23,593 bp (mouse) to 33,391 bp (chimpanzee).The most signifi cant intron sequence similarity is between human and chimpanzee and then between mouse and rat, where the introns 5, 7, and 11 are the most conserved.

Adenosine deaminases in plants -an open question
Despite the adenosine deaminase enzymes have been thoroughly described in prokaryotes, lower eukaryotes and mammals, there are only scarce reports on these enzymes in plants.Although completed genome databases of Arabidopsis thaliana and other plants show presence of putative encoding genes, the corresponding proteins have not been obtained so far nor the activity towards adenosine demonstrated.Some articles even speculate that adenosine deaminase is not present at all in plant tissue 49,50,51 or that enzyme activity is too low and the main enzyme for adenosine recycling in plants is adenosine kinase (EC 2.7.1.77) 52,53 The corresponding hypothetical plant gene product contains an adenosine/AMP conserved domain cd00443 (diff erent from cd01320 present in other ADAs) that is also present in some prokaryotic and eukaryotic enzymes annotated as ADA, adenosine/AMP deaminase or ADA- like proteins.On amino acid sequence level, putative plant enzymes show close relationship between each other, while they diff er from ADAs of other origin, especially from those of microorganisms (Tab.3).

CONCLUSIONS
Nowadays, the study of adenine/adenosine deaminases is receiving raising attention.Several active recombinant enzymes from microorganisms were prepared so far, such as ADEs from Escherichia coli 7 , Enterococcus faecalis 3 , Saccharomyces cerevisiase 54 and Schizosaccharomyces pombe 54 .These proteins have been produced mainly for biochemical and structural studies, but there is also a possible in vitro application of the recombinant proteins, i.e. in analytical chemistry.Development of a biosensor for the assay of adenine in biological fl uids that is based on adenine deaminase/xanthine oxidase/peroxidase system is in progress (Petr J, Pospíšilová H, Frébort I, Nistor M, unpublished results).
Human adenosine deaminase is very intensively investigated at the medical fi eld because of diseases induced by lack or excess of its activity.Acquired knowledge is then used in attempting to therapy the adenosine deaminasesbased illness.For examples, enzyme replacement therapy, using red blood cells as a source of encapsulated human ADA, restored both T and B cell function of patients with adenosine deaminase defi cient form of severe combined immunodefi ciency (SCID) 55 and hematopoietic stem cell gene therapy led to the successful reconstitution of immune function in a child who showed a poor response to PEG-ADA enzyme replacement 56 .ADA defi ciency was selected for the fi rst approved human gene therapy trial 57 .Determination of ADA could be used as a nonspecifi c marker of T-lymphocyte activation 58 .As a model system for human therapy, mouse is often used also in the investigation of ADA-linked disorders.Hence, murine ADA was cloned and expressed in modifi ed E. coli and monolayer cultures of murine Cl-1D LM (thymidine kinase − ) fi broblast cells derived from bone marrow stromal cells of a (C57BL/6J × C3H/HeJ)F 1 mouse and human embryonic kidney (HEK) 293 cells 59 .

1 .
A D A /A M P D , A .th a li a n a 2 .A D A /A M P D , O .s a ti v a 3 .A D A /A M P D , H . s a p ie n s 9 .A D A /A M P D , A .n id u la n s 7 .A D A /A M P D , U .m a y d is 8 .A D A /A M P D , G .z e a e 5 .A D A /A M P D , D .re ri o 4 .A D A /A M P D , B .ta u ru s 6 .A D A /A M P D , A .g a m b ia e 1 0 .A D A /A M P D , A .te rr e u s

Table 3 .
60mparison of amino acid identity between putative plant adenosine/AMP deaminases and the enzymes from other organisms containing the same conserved domain cd00443.Data were obtained using ClustalW multiple sequence alignment followed by sequence identity matrix calculation using the BioEdit software 7.0.5.360.