Orf135 from Escherichia coli is a Nudix Hydrolase Specific for

CTP, dCTP, and 5-methyl-dCTP*

Suzanne F. O’Handley‡, Christopher A. Dunn, and Maurice J. Bessman

From the Department of Biology and the McCollum-Pratt Institute,

The Johns Hopkins University, Baltimore, Maryland 21218

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 26, 2000 as Manuscript M004100200 by guest on M

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Running Title: Orf135, a CTPase and Nudix Hydrolase

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Abstract

Orf135 from E. coli is a new member of the Nudix hydrolase family of enzymes with substrate

specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been cloned for overexpression

and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-

methyl-dCTP (kcat/Km = 301,000 M-1s-1), followed by CTP (kcat/Km = 47,000 M-1s-1) and

dCTP (kcat/Km = 18,000 M-1s-1). Unlike other nucleoside triphosphate

pyrophophohydrolases of the Nudix hydrolase family discovered thus far, Orf135 is highly

specific for pyrimidine (deoxy)nucleoside triphosphates. Like other Nudix hydrolases, the

enzyme cleaves its substrates to produce a nucleoside monophosphate and inorganic

pyrophosphate, has an alkaline pH optimum, and requires a divalent metal cation for catalysis,

with magnesium yielding optimal activity. Due to the nature of its substrate specificity, Orf135

may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in controlling levels of 5-

methyl-dCTP in the cell.

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The Nudix hydrolases are a family of enzymes catalyzing the hydrolysis of substrates

consisting of a nucleoside diphosphate linked to some other moiety, x (hence the acronym

“Nudix”), and are defined by the signature sequence GX5EX7REUXEEXGU where U is a bulky

aliphatic amino acid, I, L, or V (1). The family consists of enzymes that hydrolyze (d)NTP’s (2-

8), NADH (9-10), GDP-mannose (11), ADP-ribose (10, 12-14), diadenosine polyphosphates

(10, 15-21), and diphosphoinositol polyphosphates in addition to diadenosine polyphosphates

(22-24). The Nudix hydrolases were first discovered through comparison of E. coli MutT and S.

pneumoniae MutX and from BLAST (25) searches of MutT, which revealed the signature

sequence, common in a number of open reading frames (26, 27). The family has grown to

include over 450 open reading frames in over 85 species and to include a variety of enzymes as

indicated above. Nudix hydrolases are ubiquitous throughout nature, existing in eukaryotes,

prokaryotes, and archaea (12) and appear to control the level of potentially toxic substances that

would be detrimental to the cell at elevated levels (1, 23) and to regulate the accumulation of

metabolic intermediates (1, 23).

Orf135 is a true member of the Nudix hydrolase family; it contains the signature sequence

GX5EX7REUXEEXGU and it cleaves the nucleoside diphosphate derivatives CTP, dCTP, and 5-

methyl-dCTP. Yet, it is unique in its substrate specificity, since it is the first Nudix hydrolase

highly specific towards pyrimidine substrates. In this paper, we describe the cloning and

expression of the orf135 gene, the purification and characterization of the Orf135 enzyme, and

we discuss its possible role in intermediary metabolism.

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EXPERIMENTAL PROCEDURES

Materials

Nucleic Acids- Oligodeoxynucleotides were obtained from Integrated DNA Technologies, the

plasmid pET11b was from Novagen, and chromosomal DNA from E. coli strain MG1655 was

kindly provided by Dr. Frederick R. Blattner (University of Wisconsin).

Bacterial strains- Competent cells of E. coli DH5α were obtained from Life Technologies and

E. coli HMS174(DE3) was from Novagen.

Enzymes- Pfu DNA polymerase was from Strategene, restriction enzymes NdeI and BamHI and

T4 DNA ligase were obtained from Life Technologies, and inorganic pyrophosphatase came

from Sigma.

Chemicals- IPTG was from Research Organics, Sephadex G50 from Pharmacia Biotech,

nucleotide substrates were from Sigma, and other general chemicals were from Sigma or JT

Baker.

Methods

Cloning- The orf135 gene was amplified from E. coli strain MG1655 chromosomal DNA using

the polymerase chain reaction. An NdeI restriction site was incorporated at the start of the gene,

and a BamHI site at its end using oligodeoxynucleotide primers containing these sites. The

amplified gene was purified, digested with NdeI and BamHI, and ligated into the respective

restriction sites of plasmid pET11b to place the orf135 gene under control of a T7 lac promoter

for expression. The resultant plasmid, pETorf135 was used to transform E. coli strain DH5α for

storage and E. coli strain HMS174(DE3) for expression. The sequence of orf135 in the resultant

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plasmid was confirmed using the flourescent dideoxy terminator method on a Perkin Elmer ABI

377 automated DNA sequencer by the DNA analysis facility at the Johns Hopkins University.

Expression and Enzyme Purification- E. coli strain HMS174(DE3) containing pETorf135 was

grown at 37 °C in 2 liters of broth containing 100 ug / ml Ampicilin. When growth reached an

A600 of 0.8, the culture was induced with 1mM isopropyl-β-D-thiogalactopyranoside, grown

for an additional 2 hours, and the cells (3.5 g) were harvested, washed in buffered isotonic saline

solution, recentrifuged, and stored at 80 °C.

Orf135 was extracted from the frozen cells by resuspending them in 2 volumes of buffer A

(50 mM Tris, pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol). Prior freezing was necessary to

render the protein extractable in this manner. The suspension was centrifuged, and the

supernatant (Fraction I) containing approximately 80 mg of protein was concentrated by

precipitation with 65% ammonium sulfate and dissolved in 1.2 ml buffer A (Fraction II).

Fraction II (containing approximately 65 mg of protein) was loaded onto a 1.5 x 52 cm Sephadex

G50 gel filtration column (calibrated with molecular weight standards of 14, 20, and 29 kDa) and

eluted with buffer A containing 200 mM NaCl. The fractions containing Orf135 and essentially

free of other proteins were combined (Fraction III) and concentrated by precipitation with 80%

ammonium sulfate, dissolved in buffer A to a final volume of 2 ml (Fraction IV) and stored at

–80 °C where Orf135 was stable indefinitely. Fraction IV contained 25 mg protein with 2300

units of CTPase activity.

Enzyme Assays- A standard reaction mixture of 50 ul contained 50 mM Tris-HCl, pH9, 1 mM

dithiothreital, 5 mM MgCl2, 4 mM substrate (CTP), 500 milliunits inorganic pyrophosphatase,

and 0.3 to 5 milliunits Orf135. The mixture was incubated at 37 °C for 15 minutes, stopped by

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the addition of 50 ul of 4 parts Norit (20% packed volume) and 1 part 7% perchloric acid,

centrifuged, and 50 ul of the supernatant was analyzed for inorganic orthophosphate by the

colorimetric procedure of Ames and Dubin (28). One unit of Orf135 hydrolyzes 1 µmol

substrate / min.

To identify the products and stoichiometry of the reaction, the standard reaction mixture was

scaled up 20-fold, inorganic pyrophosphatase was omitted, and the reactions were quenched

with excess EDTA (relative to magnesium). The substrate and nucleotide product were

quantitated using a high performance liquid chromatography system with a YMC ODS-AM

column, and an isocratic mobile phase of 12.5 mM citric acid, 25 mM sodium acetate, 10 mM

acetic acid, adjusted to pH 6.3 with sodium hydroxide. The substrate and nucleotide product

were detected at 254 nm and identified with standards of CTP, CDP, and CMP. Inorganic

pyrophosphate was quantitated by the colorimetric assay after hydrolysis to inorganic

orthophosphate by boiling for 15 minutes in 0.5 M HCl.

Kinetic studies were carried out using 0.1 to 4 mM substrate, 5 mM MgCl2, and 0.1 to 5

milliunits of Orf135.

Assay for mutator phenotype- orf135 was subcloned from pETorf135 and into a pTRC99A

vector containing an E. coli lac promoter, transformed into E. coli strain SB3 lacking a functional

mutT gene, and mutation frequencies were determined as described in O’Handley et al (8).

RESULTS

Subcloning, Expression, and Purification

We identified the orf135 gene, which had been sequenced and deposited as part of the E. coli

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sequencing project (29), from a BLAST search (30) of the Nudix signature sequence in MutT.

The gene was cloned directly from chromosomal DNA as described under methods and its

sequence agrees with that reported in Genbank. Expression of orf135 results in the appearance

of a major band on a denaturing polyacrylamide gel corresponding to a 15 kDa protein, not

readily detectable by eye in the same strain of E. coli containing pET11b without the insert

(Fig. 1). As has been reported for other Nudix hydrolase enzymes (8, 11, 14, 18), a majority of

the Orf135 is released into solution simply by freezing and thawing the cells, leaving most of the

other proteins behind in the cell. This results in an extract much more highly enriched in Orf135

than would result from more complete disruption of the cells such as by sonication, and it greatly

simplifies purification, requiring only an additional ammonium sulfate fractionation and gel

filtration step. This procedure yields approximately 25 mg of essentially pure enzyme from a

2 liter preparation (Fig. 1).

Orf135 migrates on a denaturing polyacrylamide gel as expected for the 15 kDa polypeptide

predicted from its amino acid composition, and it elutes from a G50 gel filtration column as

expected for a monomer of this size.

Substrate Specificity

Like MutT and Orf17, Orf135 is a (deoxy)ribonucleoside triphosphatase; however, the

substrate specificity is markedly different for these three enzymes. Whereas MutT and Orf17

hydrolyze all 8 canonical nucleoside triphosphates with preference for dGTP and dATP,

respectively (2, 8), Orf135 is very specific for CTP, dCTP, and 5-methyl-dCTP (Table I). This

is the first (deoxy)ribonucleoside triphosphatase of the Nudix hydrolase family that is base

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specific and the first Nudix hydrolase in general that is specific for pyrimidine substrates (1).

Orf135 hydrolyzes UTP and dTTP, at less than 5% of the rate of CTP, and there is no significant

hydrolysis of ATP, dATP, GTP, or dGTP. Also, as we have observed with MutT and Orf17,

cleavage of the pyrophosphate linkage in the nucleoside diphosphates by Orf135 is minimal and

cleavage of the nucleoside monophosphates by Orf135 is undetectable.

The influence of the various functional groups of the substrates on the rate of hydrolysis is

compared in Fig. 2. 5-methyl-dCTP and CTP have an amino group at the C4 position which is

replaced in dTTP and UTP by a keto group. 5-methyl-dCTP and CTP are hydrolyzed at rates

50 times greater than are dTTP and UTP, respectively, indicating the importance of the C4 amino

group for recognition and catalysis. Likewise, 5-methyl-dCTP is hydrolyzed more rapidly than

dCTP and dTTP at a higher rate than dUTP, both by a factor of approximately 5, indicating that

the 5-methyl group also enhances the overall rate, although not as dramatically as the influence

of the C4 amino group. As for the sugar, the presence of the hydroxyl group at the C2’ position

does not have that great of an effect on activity (the rate of hydrolysis of CTP is double that of

dCTP), but epimerization of the hydroxyl group at the C2’ position decreases activity by

approximately 20 fold as indicated by comparison of cleavage of CTP versus ara-CTP. It will

be interesting to examine whether these important functional groups on the substrates make vital

contacts with amino acids in the protein by analyzing a 3-dimensional structure of the enzyme

complexed with the substrate.

Kinetic analysis of the preferred substrates (Table II) shows that Orf135 cleaves 5-methyl-

dCTP with a catalytic efficiency 6 times greater than that for CTP and 17 times greater than that

for dCTP. The possible biological significance of these differences will be discussed below.

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Requirements of the Enzyme

pH optimum- Orf135 has optimal activity between pH 8.5 and 9.0 in Tris–HCl or glycine

buffers with the rate dropping to 50% at pH 7.8 and 9.8. This alkaline pH optimum is typical of

the Nudix hydrolase family of enzymes (with the pH optima for MutT, Orf17, Orf257, Orf186,

and Orf1.9 being pH 9.0, 8.6, 8.5, 9.3, and 9.0, respectively (2, 8-11)), indicating a common

mechanism of hydrolysis as discussed previously in O’Handley et al. (10).

divalent cation requirement- As with all of the other Nudix hydrolases discovered thus far,

Orf135 absolutely requires a divalent metal cation for activity. Magnesium is most effective

with optimal activity at 5 mM Mg+2 in the presence of 4 mM substrate. Mn+2 can partially

substitute for Mg+2 with approximately 10% activity at optimal concentration, while no activity

was observed in the presence of Zn+2, Co+2, or Ca+2.

Product Formation

The products of the hydrolysis of CTP were determined from a scaled up, standard reaction

excluding inorganic pyrophosphatase. The decrease of CTP and increase of the nucleotide

product, CMP, during the course of the reaction were monitored over time by chromagraphic

analysis as shown in Fig. 3. The other product formed, inorganic pyrophosphate, was quantified

by the colorimetric assay of Ames and Dubin (28) after hydrolysis to inorganic orthophosphate

as described under methods, and this is also shown in Fig 3. The CTP hydrolyzed corresponds to

the CMP and inorganic pyrophosphate produced in a 1:1 molar ratio at each time point. No CDP

was detected throughout the reaction, and no inorganic orthophosphate appeared as analyzed by

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the method of Fiske and Subbarrow (31), which is specific for inorganic orthophosphate and will

not detect pyrophosphate. Thus the hydrolysis of CTP catalyzed by Orf135 may be written as

follows:

CTP + H2O → CMP + PPi

REACTION 1

The reaction products are similar to those generated by the other nucleoside triphosphate

pyrophosphohydrolases of the Nudix hydrolase family. MutT and MutX hydrolyze dGTP to

dGMP and pyrophosphate (2, 7) and Orf17 hydrolyzes dATP to dAMP and pyrophosphate (8).

Using H218O, we have shown that both MutT and Orf17 hydrolyze their respective substrates by

nucleophylic attack at the β phosphorus (8, 32). Since Orf135 yields similar products, it most

likely uses a similar mechanism to hydrolyze CTP.

DISCUSSION

As can be seen from a list of the known Nudix hydrolase enzymes, Orf135 fits in well as a

member of the Nudix hydrolase family (Fig. 4). All of the other enzymes are distinct from

Orf135, but they all share the common signature sequence, GX5EX7REUXEEXGU (where U is

I, L, or V), characteristic of this family (1), and they all hydrolyze nucleoside diphosphate

compounds.

A BLAST search using Orf135 as the query identifying many of these previously discovered

Nudix hydrolases, as well as a number of unknown open reading frames, is shown in Fig. 5. The

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closest matches to identified enzymes are to MutT from P. vulgeris, H. influenzae, and E. coli,

with E values of 5 x 10-10, 3 x 10-9, and 8 x 10-8, respectively. Also, when threading programs

were used to compare the predicted structures of Orf135 and the other Nudix hydrolases to the

solved NMR solution structure of MutT, Orf135 had the highest match of any of the known,

characterized Nudix hydrolases (results not shown). However, as has been described throughout

this paper, Orf135 is distinct from MutT in its substrate specificity, preferentially hydrolyzing

CTP, dCTP, and 5-methyl-dCTP as opposed to dGTP. Because Orf135 appears more similar to

MutT in primary and predicted secondary structure than other Nudix hydrolases, it was important

to ascertain whether the cellular roles of Orf135 and MutT are distinctly different from one

another. Accordingly, a plasmid carrying the orf135 gene was transformed into a mutT- strain

of E. coli, and analyzed for a decrease in mutation frequency. Orf135 did not complement MutT

(results not shown). This result was not surprising to us, since no other Nudix hydrolases except

MutT orthologs active on dGTP have been shown to complement MutT (8, 12, and personal

observations), and it demonstrates that even open reading frames with similar primary and

(predicted) secondary structures can have different enzymatic activities and cellular functions.

Thus, as we have pointed out (1), caution must be exercised when analyzing information from

BLAST searches and databases of proteins containing the Nudix signature sequence. For

example, the Japanese E. coli sequencing project lists Orf135 (accession # BAA15549) as a

mutator MutT protein or dGTP pyrophosphohydrolase, whereas the report for Orf135 (accession

# AAC74829), deposited by Blattner’s group (29) as part of their sequencing of E. coli, simply

states that Orf135 is 37% identical to 125 residues of MutT from H. influenzae.

What then is the possible function of Orf135 in the cell? The best substrates discovered for

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Orf135 in vitro are CTP, dCTP, and 5-methyl-dCTP. The role of Orf135 may be to regulate the

levels of these compounds or other cytidine triphosphate analogs in the cell as discussed below.

Methylation of the C5 position of cytosine by DNA (cytosine-5-) methyltransferase is

involved in the differential control of gene expression (33). As a result of DNA degradation, 5-

methyl-dCMP is produced. In human cells, nucleoside monophosphate kinase does not appear

to recognize 5-methyl-dCMP as a substrate, and thus phosphorylation of 5-methyl-dCMP to

5-methyl-dCTP does not occur (34, 35). It has been suggested that this prevents 5-

methylcytosine from being randomly incorporated into DNA, since 5-methyl-dCTP is an

excellent analogue of dCTP and can replace it completely (36). No similar studies have been

reported for E. coli, and so the significance of 5-methyl-dCTPase here is moot. There are

however bacteriophages which induce kinases that can phosphorylate 5-methyl-dCMP (37) and

5-hydroxymethyl-dCMP (38), and Orf135 could be part of a defense mechanism against

infection.

Furthermore, Orf135 may monitor the intracellular accumulation of CTP, a key metabolite in

both pyrimidine and lipid biosynthesis. Orf135 may play a role in regulating the synthesis of

pyrimidines in E. coli by hydrolyzing CTP. CTP is the negative regulator of the entire

pyrimidine biosynthetic pathway in E. coli; binding to aspartate transcarbamoylase, the

committed step in the synthesis of UTP and CTP (39). If CTP accumulates, its inhibition of

aspartate transcarbamoylase not only inhibits its own synthesis, but also the synthesis of UMP,

UDP, and UTP. Since dTMP is synthesized from other pyrimidine deoxyribonucleotides with

the common pyrimidine precursor being UMP, the synthesis of dTMP would also be inhibited by

CTP accumulation. This would ultimately affect DNA synthesis. Orf135 may circumvent these

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effects through hydrolysis of CTP. It is interesting to note that dATP, the major negative

regulator of the deoxynucleotide biosynthetic pathway, is the substrate of another Nudix

hydrolase, Orf17 from E. coli, which hydrolyzes it to dAMP and pyrophosphate (8).

In lipid biosynthesis, CTP is utilized in the synthesis of CDP-ethanolamine and CDP-

diacylglycerol, both important intermediates in the synthesis of glycerophospholipids, which are

major components of cell membranes (40). There have been several studies which show a direct

correlation between CTP levels and phospholipid biosynthesis. Overexpression of CTP

synthetase (41) or expression of a CTP synthetase mutant less sensitive to inhibition by CTP

(42), causes an increase in CTP levels, leading to an increase in phospholipids and neutral lipids.

On the other hand, when cyclopentenylcytosine, a potent and specific inhibitor of CTP

synthetase was added to cells, the pool size of CTP dropped to approximately 10% of controls,

and phospholipid biosynthesis was re-routed away from CTP utilizing reactions towards neutral

lipid biosynthesis (43). As pointed out by Hatch and McClarty in the latter paper, “the cellular

CTP level may be a universal signal or switch for all phospholipid biosynthesis”. Thus, the

regulation of CTP levels is vital to the cell, and Orf135 may play a role in this regard.

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Footnotes

* This work was supported by National Institutes of Health Grant GM 18649. This is publication

XXXX from the McCollum-Pratt Institute. The costs of publication of this article were defrayed

in part by the payment of page charges. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ Present address: Department of Chemistry, University of Richmond, Richmond, VA 23173.

To whom correspondence should be addressed: Tel.: 804-289-8245; FAX: 804-287-1897;

E-mail: sohandle@richmond.edu

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Figure Legends

Figure 1. Expression and purification of the Orf135 protein. A 15% polyacrylamide gel

containing 1% SDS contained the following: lane 1, reference proteins with molecular weights

of 66, 45, 36, 29, 24, 20, 14, and 6.5 kDa. lane 2, crude extract from cells containing pET11b.

lane 3, crude extract from cells containing pETorf135. lane 4, Fraction I, supernatant obtained

from freezing and thawing the cells. lane 5, Fraction IV obtained from purification over a G50

column and concentration of pulled fractions. Lanes 3-5 each contain 3 µg protein. Lane 2

contains a comparable amount of crude extract as that in lane 3.

Figure 2. Contribution of substrate functional groups to activity by Orf135. The relative

activities of Orf135 on the various ribo- and deoxyribonucleoside triphosphates is shown below

each base.

Figure 3. Products of the reaction of Orf135 with CTP. The reaction was carried out as

described under “experimental procedures”. The decrease in CTP (•) and increase in CMP (o)

were monitored and quantified by high performance liquid chromatography. Inorganic

pyrophosphate (♦) was hydrolyzed to inorganic orthophosphate with HCl and quantified by the

colorimetric assay as described under experimental procedures.

Figure 4. The Nudix hydrolase enzymes. Each enzyme and its source is listed along with the

nudix box or signature sequence and the associated activity. The conserved amino acids are

bolded. Numbers in parentheses are published references

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Figure 5. Results of a recent BLAST search of Orf135. The entire sequence of Orf135 was

searched by BLAST against a number of nonredundant data bases (GenBank CDS translations,

PDB, SwissProt, PIR, and PRF). The 49 best matches are listed in order of identity and

similarity to Orf135. Of the Nudix hydrolases with known activities, the 3 best matches to

Orf135 are MutT enzymes. The Nudix sequences for each are shown with the conserved amino

acids bolded. Those open reading frames with identified activities are listed in the second

column. Their activities (except those marked by asterisks) can be found in Fig. 4. Those

marked with an asterisk have been found to have enzymatic activity but have not been

completely characterized as yet.

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N

N

NH2

O

NH

N

O

O

NH

N

O

O

CH3N

N

NH2

CH3

O

C U T 5MeC

ribose:

deoxyribose:

48%

23%

1%

<1% 2% 100%

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0

20

40

60

80

100

0 5 10 15

CT

P, C

MP

, or

PP

i (nm

ol)

Time (min)

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Enzyme Signature Sequence Activity Reactions

Orf135 (E.coli) GKVEPDESQRQALVRELREELGI ♥MutT (E.coli) GKIEMGETPEQAVVRELQEEVGI ♣ (2)

MutX (S.pneumoniae) GKLERGETPQECAVREILEETGL ♣ (7)

MutT (P.vulgaris) GKLEDNETPEQALLRELQEEIGI ♣ (6) ♥ (5Med)CTP + H2O → (5Med)CMP + PPi

MutT (H.sapiens) GKVQEGETIEDGARRELQEESGL ♣ (3)

MutT (M.musculus) GKVQEGETIEDGAKRELLEESGL ♣ (4) ♣ (8oxo)dGTP + H2O → (8oxo)dGMP +PPi

MutT (R.norvegicus) GKVQEGETIEDGAKRELLEESGL ♣ (5)

Orf17 (E.coli) GSVEEGETAPQAAMREVKEEVTI ♠ (8) ♠ dATP + H2O → dAMP + PPi

Orf257 (E.coli) GFVEVGETLEQAVAREVMEESGI • (9)

Orf186 (E.coli) GLIDPGESVYEAANRELKEEVGF *•# (10) • NADH + H2O → AMP +NMN

MJ1149 (M.jannaschii) GFVECGETVEEAVVREIKEETGL * (12)

Slr0787 (Synechocystis sp.) GFIKQNETLVEGMLRELKEETRL * (13) * ADP-ribose + H2O → AMP + ribose-5-P

Orf209 (E.coli) GMLEEGESVEDVARREAIEEAGL * (14)

YSA1 (S. cereviciae) GLIDAGEDIDTAALRELKEETGY * (14) ♦ GDP-mannose+H2O → GDP+mannose

YQKG (B. subtilus) GKLEKGEEPEYTALRELEEETGY * (14)

YZZG (H. influenzae) GMVEKGEKPEDVALRESEEEAGI * (14) # Ap3A + H2O → AMP + ADP

Orf1.9 (E.coli) GRVQKDETLEAAFERLTMAELGL ♦ (11)

Ap4Aase (H.sapiens) GHVEPGEDDLETALRETQEEAGI λ (15) λ Ap4A + H2O → AMP + ATP

Ap4Aase (S.scrofa) GHVEPGESDLQTALRETQEEAGI λ (16)

Ap4Aase (L.angustifolius) GGIDEGEDPRNAAIRELREETGV λ (17) ∆ Ap6A + H2O → ADP + Ap4IalA (B. bacilliformis) GGIDEGEEPLDAARRELYEETGM λ (18,19)

YA9E (S. pombe) GGWEADESVQQAALREGWEEGGL ∆ (20) $ PP-InsP6 + H2O → P-InsP6 + Pi

YOR163w (S. cereviciae) GVEKDEPNYETTAGRETWEEAGC ∆ (21)

DIPP (H.sapiens) GGMEPEEEPSVAAVREVCEEAGV ∆$ (22-24)

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Organism Enzyme Accession or ID # Nudix Sequence E. coli Orf135 AAC74829 GKVEPDESQRQALVRELREELGIM. tuberculosis CAB09019 GKVAAGETERAALARELAEELGLSynechocystis sp. BAA16660 GKLEPGETAAECIVREVREEIAIP. vulgaris MutT P32090 GKLEDNETPEQALLRELQEEIGIH. influenzae MutT P44932 GKVDAGETPEQALKRELEEEIGIE. coli MutT P08337 GKIEMGETPEQAVVRELQEEVGIM. thermoautotrophicum 2621161 GKIGTGESLEEALKREVKEETNLM. thermoautotrophicum 2622443 GKVRAGETLDEALSREVREETGLS. ambofaciens P32091 GVLELDETPETGVAREVWEETGIB. subtilis BAA19270 GRVDPGESAEEAAVREILEETGYB. subtilis CAB08056 GKMESGESVRDSVIREYREETGIS. coelicolor CAA19915 GVLELDETPEAGVAREVWEETGISynechocystis sp. BAA18435 GKVEWGETLEAALKREFQEEVGLE. coli Orf141* P52006 GGVEPGERIEEALRREIREELGEE. coli Orf153* 1787379 GHLEADETLVEAAARELWEETGIA. aeolicus 2982891 GNIEPGEKPEETAVREVWEETGVS. anulatus CAA63159 GAVEDGETHHEALAREIAEETGWM. jannaschii MJ1149 D64443 GFVECGETVEEAVVREIKEETGLA. thaliana 1871177 GYLEVGESAAQGAMRETWEEAGAP. aeruginosa 3549119 GFVEAGESVEQCVVREVREEVGVM. xanthus AAD34635.1 GRLEAGESPAQAAARELEEETGLH. sapiens Ap4Aase NP 001152.1 GHVEPGEDDLETALRETQEEAGIM. leprae CAB164501 GKVDPGETAPMAAVREVFEETGHM. musculus Ap4Aase P56380 GHVDPGENDLETALRETREETGIB. thiaminolyticus YZGD* P46351 GHVERGESVEEAIVREIREETGLH. influenzae Q57045 GGINDNESAEQAMYRELHEEVGLM. tuberculosis CAB06583 GARDSHETPEQTAVRESSEEAGLE. coli Orf176* Q46930 GGINPGESAEQAMYRELFEEVGLC. elegans CAB04835 GRVEAGETIEEAVVREVKEETGYS. cerevisiae 870734 GKISKDENDIDCCIREVKEEIGFH. sapiens DIPP 3978224 GGMEPEEEPSVAAVREVCEEAGVS. pneumoniae MutX P41354 GKLERGETPQECAVREILEETGLS. scrofa Ap4Aase P50584 GHVEPGESDLQTALRETQEEAGIP. aerophilum 4099062 GNVELGETPEQAALREIKEETGLS. coelicolor CAA16467 GHVEEGETLLEALAREVEEETGWB. subtilis 2293161 GKVEPMECAEEAALREVKEETGAA. pernix BAA81091.1 GHVRLGETLEEVAARELEEETGIE. coli Orf257 P32664 GFVEVGETLEQAVAREVMEESGIP. aeruginosa AAD22458.1 GGINDRETPEEALYRELNEEVGLT. maritima AAD36256.1 GKLDPGESPEECAKRELEEETGYA. pernix BAA81066.1 GRVEYSESIPLCLVREMKEEAGIS. coelicolor CAA18515 GFVRDGEDLAQAAARELAEETGLS. coelicolor CAA19392 GGVEGDETRAEAARRELLEETGIH. thermoluteolus BAA76604.1 GHVEPGETLVAAVVRETLEETRFM. musculus MutT P53368 GKVQEGETIEDGAKRELLEESGLS. coelicolor CAA20803 GVVEDGEDVAVAAARELEEETGWS. pombe CAB39798.1 GFLEPGESLEEAVVRETYEESGVH. sapiens AAD01636.2 GLSEPEEDIGDTAVREVFEETGIE. coli Orf17 P24236 GSVEEGETAPQAAMREVKEEVTI

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Table I

Substrate specificity of Orf135

All substrates were present at a concentration of 4 mM and were assayed usingthe colorimetric procedure described in "Methods", except for CDP-ethanolamine,CDP-choline, CDP-glucose, and CDP-glycerol, which were assayed as describedin OHandley et al. (10).

SubstrateaSpecific

activitya

Relative

activity

units mg-1 %

5methyl-dCTP 193 (100)

CTP 93 48

dCTP 45 23

araCTP 5 3

dTTP 4 2

UTP 1 1

dUTP < 1 < 1

CDP 3 2

dCDP 2 1

CMP < 1 < 1

dCMP < 1 < 1

(d)ATP, (d)GTP < 1 < 1

CDP-ethanolamine, CDP-choline,

CDP-glucose, CDP-glycerol

< 1 < 1

aA unit of enzyme hydrolyzes 1µmol of substrate per min.

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Table II

Kinetic parameters for Orf135

The standard assay as described under “Methods” was used with substrate concentrations of 0.1 to 4 mM for all. KM and Vmax were determined from a non-linear regression analysis (44) and kcat was calculated from Vmax. A unit of enzymehydrolyzes 1 µmol of substrate per min.

Substrate Vmax kcat Km kcat/Km

units mg-1 s-1 mM 104 M-1 s-1

5methyl-dCTP 206±6 51.7±1.5 0.172±0.034 30.1±4.4

CTP 103±4 25.8±1.0 0.551±0.076 4.7±1.3

dCTP 57±2 14.3±0.5 0.767±0.083 1.8±0.6

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Suzanne F. O'Handley, Christopher A. Dunn and Maurice J. Bessman5-methyl-dCTP

Orf135 from Escherichia coli is a Nudix Hydrolase Specific for CTP, dCTP, and

published online October 26, 2000J. Biol. Chem. 

  10.1074/jbc.M004100200Access the most updated version of this article at doi:

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