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PubMed2026

The Absence of E. coli Nucleoid-Associated Protein FIS at Low Temperature Leads to an Adaptation Response That Causes a Shift Towards Genome Compaction in Small Rods.

In contrast to the rod shape at 37°C, the morphology of Escherichia coli cells at temperatures just above the minimum temperature of growth is small rods. A study was initiated to determine the requirement of nucleoid-associated protein FIS for growth and genome compaction in the small rods at low temperature. Growth and nucleoid staining analyses revealed that the fis null mutant displayed decreased growth and initially formed filaments containing decondensed nucleoids at 12°C, indicating that FIS facilitates production of small rods with condensed nucleoids at low temperature. However, characterized by biphasic growth at low temperature, the fis null mutant exhibited increased growth, cell division, and nucleoid condensation following an acclimation phase. Therefore, the absence of FIS with nucleoid decondensation leads to an adaptation mechanism, termed FIS Null Adaptation Response, that causes a shift towards nucleoid condensation resulting in genome compaction in small rods. Furthermore, overproduction of the HsIVU protease suppressed the cold-sensitive phenotypes of the fis null mutant indicating that degradation of a natural substrate of the protease alleviates the requirement of FIS at low temperature. In addition, null mutations of genes encoding natural substrates of HsIVU (exoribonuclease RNAse R, and cell division inhibitor SulA) were identified as extragenic suppressors of the fis null mutation.

Escherichia coli↗
PubMed2006

Polynucleotide phosphorylase negatively controls spv virulence gene expression in Salmonella enterica.

Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.

Animals↗
PubMed1988

Cloning, characterization, and effects of overexpression of the Escherichia coli rnd gene encoding RNase D.

RNase D is a 3'-exoribonuclease whose in vitro specificity has suggested that it is involved in the processing of tRNA precursors. Its in vivo role has remained unclear, however, because mutant cells devoid of the enzyme display no defect in growth or tRNA processing. To learn more about the structure and function of RNase D, we cloned the Escherichia coli rnd gene, which is thought to code for this enzyme. The rnd gene was isolated from a cosmid library based on elevated RNase D activity and was subcloned as a 1.4-kilobase-pair fragment in pUC18. Maxicell analysis of the cloned fragment revealed that a single protein of approximately 40 kilodaltons, which is the size of RNase D, was synthesized. The rnd gene is present as a single copy on the E. coli chromosome and is totally absent in a deletion mutant. Cells that harbored the cloned rnd gene displayed RNase D activity that was elevated as much as 20-fold over that of the wild type. As growth of the culture progressed, however, RNase D specific activity declined dramatically, together with a similar decrease in plasmid copy number. In contrast, no decrease in copy number was observed with an inactive rnd gene. Placement of the rnd gene downstream from the lac promoter led to inducible RNase D overexpression and concomitantly slowed cell growth. These findings support the idea that rnd is the structural gene for RNase D and indicate that elevated RNase D activity is deleterious to E. coli.

Cloning, Molecular↗
PubMed2004

Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: Processing of pnp transcripts in Streptomyces antibioticus.

We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.

Amino Acid Sequence↗
PubMed2004

Bacillus subtilis YhcR, a high-molecular-weight, nonspecific endonuclease with a unique domain structure.

In a continuing effort to identify ribonucleases that may be involved in mRNA decay in Bacillus subtilis, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3'-to-5' exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken. These experiments revealed the presence of a high-molecular-weight nuclease encoded by the yhcR gene that was active in the presence of Ca(2+) and Mn(2+). YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3'-monophosphate products, similar to the well-characterized micrococcal nuclease of Staphylococcus aureus. YhcR appears to be located principally in the cell wall and is likely to be a substrate for a B. subtilis sortase. Zymogram analysis suggests that YhcR is the major Ca(2+)-activated nuclease of B. subtilis. In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named "NYD," for "new YhcR domain."

Amino Acid Sequence↗
PubMed2005

Function of the conserved S1 and KH domains in polynucleotide phosphorylase.

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.

Binding Sites↗
PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗
PubMed2006

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Coronaviridae↗
PubMed2006

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines.

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.

3' Untranslated Regions↗
PubMed2006

Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis.

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-beta-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

Adenosine Triphosphate↗
PubMed2006

A new function in translocation for the mitochondrial i-AAA protease Yme1: import of polynucleotide phosphorylase into the intermembrane space.

Polynucleotide phosphorylase (PNPase) is an exoribonuclease and poly(A) polymerase postulated to function in the cytosol and mitochondrial matrix. Prior overexpression studies resulted in PNPase localization to both the cytosol and mitochondria, concurrent with cytosolic RNA degradation and pleiotropic cellular effects, including growth inhibition and apoptosis, that may not reflect a physiologic role for endogenous PNPase. We therefore conducted a mechanistic study of PNPase biogenesis in the mitochondrion. Interestingly, PNPase is localized to the intermembrane space by a novel import pathway. PNPase has a typical N-terminal targeting sequence that is cleaved by the matrix processing peptidase when PNPase engaged the TIM23 translocon at the inner membrane. The i-AAA protease Yme1 mediated translocation of PNPase into the intermembrane space but did not degrade PNPase. In a yeast strain deleted for Yme1 and expressing PNPase, nonimported PNPase accumulated in the cytosol, confirming an in vivo role for Yme1 in PNPase maturation. PNPase localization to the mitochondrial intermembrane space suggests a unique role distinct from its highly conserved function in RNA processing in chloroplasts and bacteria. Furthermore, Yme1 has a new function in protein translocation, indicating that the intermembrane space harbors diverse pathways for protein translocation.

ATP-Dependent Proteases↗
PubMed1996

Prions and RNA viruses of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is host to the dsRNA viruses L-A (including its killer toxin-encoding satellite, M) and L-BC, the 20S and 23S ssRNA replicons, and the putative prions, [URE3] and [PSI]. review the genetic and biochemical evidence indicating that [URE3] and [PSI] are prion forms of Ure2p and Sup35p, respectively. Each has an N-terminal domain involved in propagation or generation of the prion state and a C-terminal domain responsible for the protein's normal function, nitrogen regulation, or translation termination, respectively. The L-A dsRNA virus expression, replication, and RNA packaging are reviewed. L-A uses a -1 ribosomal frameshift to produce a Gag-Pol fusion protein. The host SK12, SK13 and SK18 proteins block translation of nonpoly(A) mRNAs (such as viral mRNA). Mutants deficient in 60S ribosomal subunits replicate L-A poorly, but not if cells are also ski-. Interaction of 60S subunits with the 3' polyA is suggested. SKI1/XRN1 is a 5'--> 3' exoribonuclease that degrades uncapped mRNAs. The viral Gag protein decapitates cellular mRNAs apparently to decoy this enzyme from working on viral mRNA.

Amino Acid Sequence↗
PubMed2006

Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3'hExo/ERI-1 family of 3' to 5' exonucleases.

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.

Amino Acid Sequence↗
PubMed2005

Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export.

Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2004

Functional analysis of mRNA scavenger decapping enzymes.

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.

Base Pairing↗
PubMed2004

Nematode m7GpppG and m3(2,2,7)GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS.

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.

Animals↗
PubMedDate not supplied

[Age-dependent gene induction in the quail oviduct. Role of post-transcriptional changes in the aging process].

In 1974 we introduced the quail oviduct system to molecular gerontology as a model for investigating age-dependent gene realization at the molecular level. During ageing the amounts of synthesized ovalbumin and avidin, the two major oviduct proteins, decrease to reach almost 0-values. Soon it emerged that the reasons for the reduced ovalbumin and avidin syntheses were not only on the transcriptional but in addition on the posttranscriptional level. It was found that the observed reduced capacity for protein biosynthesis was an age-correlated impairment of those enzyme systems which are involved in the processing of hnRNA to mRNA and in the transport of mRNA from nucleus to cytoplasm. After having firstly discovered and purified some key enzymes of mRNA processing ( endoribonucleases IV, V and VII; U1- snRNP -associated endoribonuclease; poly(A)-specific exoribonuclease ), and secondly studied in detail the enzymically controlled nucleocytoplasmic mRNA transport system, we observed that polyadenylation and perhaps also splicing of hnRNA (two processes in posttranscriptional modification of mRNA) are impaired during ageing. Pronounced changes occur especially at the level of polyadenylation of hnRNA; analytic and enzymic studies revealed that, in parallel with ageing, poly(A) catabolism increases although poly(A) anabolism remains unchanged. The consequences of this change are: reduced stability of mRNA, probably reduced splicing efficiency, reduced binding of mRNA to the inner nuclear membrane or lamina, and reduced nucleocytoplasmic transport of mRNA. The data suggest that changes at the posttranscriptional level are as important in ageing as changes in transcription, and they may explain the observed, age-dependent reduction of the amount of functional mRNA in the cytoplasm.

Aging↗
PubMedDate not supplied

[Use of a TLC method for the simultaneous determination of an enzyme complex of nucleic acid metabolism during the isolation of polynucleotide phosphorylase].

For the simultaneous determining of several enzymes of nucleic acid metabolism during polynucleotide phosphorylase isolation TLC was used. It was found that using TLC one can simultaneously detect six and more enzymes, e. g. polynucleotide phosphorylase, 5'-nucleotidase, exoribonuclease together with nucleosidediphosphatase, desaminase etc. The method is simple and accessible.

5'-Nucleotidase↗