Báo cáo khoa học: Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection ppt

One predicted class IIIa and six predicted class IIIc mycobacterial cyclase genes contain variations at canonical positions of the Keywords Adenylyl cyclase; cyclic nucleotide; guanylyl

tuberculosis H37Rv uses a novel mode for substrate

selection

Lucila I Castro, Corinna Hermsen, Joachim E Schultz and Ju¨rgen U Linder

Abteilung Pharmazeutische Biochemie, Fakulta¨t fu¨r Chemie und Pharmazie, Universita¨t Tu¨bingen, Germany

The second messenger cAMP is synthesized by a large

variety of adenylyl cyclases (ACs) which are separated

into five classes that are not related in their protein

sequences [1–3] The vast majority of ACs belongs to

class III which recently has been subdivided into

clas-ses IIIa to IIId [4] The catalytic domain of class III

ACs has been termed cyclase homology domain

(CHD) and appears to be linked with different protein

domains which in several instances have been shown

to impart peculiar regulatory features [5] (reviewed in

[4]) So far, all CHDs operate as dimers with the

cata-lytic centre positioned at the dimer interface [6–8]

Based on mutational and structural data catalysis is

thought to rest on six highly conserved residues which

are spaced in register in the CHDs Two aspartate

resi-dues coordinate two metal ions (Mg2+ or Mn2+), an

asparagine and an arginine stabilize the transition-state

and a lysine-aspartate couple specifies ATP as a sub-strate [9–11] A common variant to this canon is the exchange of the usual substrate specifying aspartate for a threonine or serine in class IIIb ACs [4] The hydroxyl group specifically serves as a hydrogen-bond acceptor and in this respect has the same function as the canonical aspartate [12,13] However variations in all six canonical catalytic residues do occur as evident from whole genome sequencing projects [4] and the functional consequences of such changes are just beginning to be understood [14]

The genome of the human pathogen Mycobacterium tuberculosis H37Rv contains 15 ORFs that code for CHDs [15] Two belong to class IIIa, four to class IIIb and nine to class IIIc [4] One predicted class IIIa and six predicted class IIIc mycobacterial cyclase genes contain variations at canonical positions of the

Keywords

Adenylyl cyclase; cyclic nucleotide; guanylyl

cyclase; Mycobacterium tuberculosis;

substrate specificity

Correspondence

J U Linder, Abteilung Pharmazeutische

Biochemie, Fakulta¨t fu¨r Chemie und

Pharmazie, Universita¨t Tu¨bingen,

Morgenstelle 8, 72076 Tu¨bingen, Germany

Fax: +49 7071 295952

Tel: +49 7071 2974676

E-mail: juergen.linder@uni-tuebingen.de

(Received 27 March 2005, revised 13 April

2005, accepted 18 April 2005)

doi:10.1111/j.1742-4658.2005.04722.x

Class III adenylyl cyclases usually possess six highly conserved catalytic residues Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes This suggests that a variety of catalytic mechanisms may actually exist The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine The recombin-ant adenylyl cyclase domain was active with a Vmax of 8 nmol cAMPÆ

mg)1Æmin)1 Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspar-tate consensus abolished activity This argues for a novel mechanism of substrate selection which depends on two noncanonical residues Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases

Abbreviations

AC, adenylyl cyclase; CHD, cyclase homology domain; GC, guanylyl cylase; HTH, helix-turn-helix; PDE, cyclic nucleotide phosphodiesterase.

catalytic centre [4] The four class IIIb cyclases contain

the threonine variant mentioned above To date almost

all canonical and all class IIIb mycobacterial ACs

have been investigated (class IIIa: AC Rv1625c; class

IIIb: Rv1318c, Rv1319c, Rv1320c, Rv3645; class IIIc:

Rv1264, Rv1647) [8,16–19] Of the noncanonical

CHDs only Rv1900c (class IIIc) has been examined in

detail [14] Here, the substrate-specifying

lysine-aspar-tate pair is replaced by asparagine-asparlysine-aspar-tate and the

catalytic asparagine is altered to histidine Structure

determination and mutagenesis experiments

demon-strated that Rv1900c adopts a mode of catalysis in

which these three otherwise canonical residues are

dis-pensable

We investigated the mycobacterial Rv0386 gene

product as a class IIIc AC which has a

glutamine-asparagine pair at the positions defining ATP as a

sub-strate instead of the lysine-aspartate consensus We

show that the purified catalytic domain of Rv0386 is

active as an AC which has an unusually high GC

side-activity Mutational analysis of Rv0386 demonstrated

that the catalytic activity specifically depends on

the noncanonical glutamine-asparagine couple This

strongly indicates that an alternative substrate-binding

mechanism evolved in Rv0386, distinct from that in

canonical ACs A model of purine-binding in Rv0386

is proposed

Results

Sequence analysis

The M tuberculosis gene Rv0386 codes for a

multido-main protein of 1085 amino acids (117 kDa, Fig 1A)

An AC catalytic domain is located at the N-terminus (amino acids 1–167), which is characterized as a class IIIc CHD because of a shortened ‘arm’ region [4] Strikingly the canonical substrate-defining residues, lysine-aspartate, correspond to Gln57 and Asn106 in Rv0386, respectively (Fig 1B, [4]) Therefore it was not at all a forgone conclusion whether the CHD of Rv0386 would in fact display AC activity

Further, sequence analysis by SMART and INTER-PRO-scan showed that the CHD is linked via 12 amino-acid residues to an AAA-ATPase domain (NB-ARC type [20], amino acids 180–477), a tetratrico-peptide repeat (TPR)-like region (amino acids 646–968) and a C-terminal helix-turn-helix (HTH) DNA-binding domain (amino acids 1024–1081, luxR family [21]) An identical domain composition, i.e a CHD linked in this order to these three domains is present in the putative

AC genes Rv1358 and Rv2488c from M tuberculosis Moreover, the AAA-ATPase⁄ NB-ARC domain is sim-ilar to the respective domains of several bacterial tran-scriptional regulators (e.g 40% similarity to afsR of Streptomyces coelicolor [22]) Therefore the presence of the HTH DNA-binding domain strongly suggests that

in Rv0386 an AC may be functionally linked with a transcriptional regulator

AC activity of the Rv0386 CHD

We expressed the CHD of Rv0386 (amino acids 1–175) in Escehrichia coli as a soluble protein and purified it to homogeneity (Fig 2) At 4.9 lm recom-binant Rv0386(1)175) displayed an AC activity of 5.0 nmol cAMPÆmg)1Æmin)1 with Mn2+ as a metal cofactor (Table 1) Activity with Mg2+ was below the

D

H

C A A N A B - A - A T R P C a e /

7 7 4 0

8 1

6 8 3 0 v R

) s d i c o i m a 5 0 (

e k i -R P T

6 4

1 8 0 1 4 2 0 1

H T H ) R x u l (

A

B

Fig 1 Sequence analysis (A) Domain com-position of Mycobacterium tuberculosis Rv0386 (B) Local alignment of Rv0386 with the noncanonical class IIIc AC Rv1900c, the canonical class IIIc AC Rv1264 and the canonical class IIIa AC Rv1625c from M.tuberculosis The six residues implicated

in catalysis by canonical ACs are boxed.

a, adenine-specifying; m, metal-coordinating;

c, catalytic transition-state stabilizing Solid arrowheads mark the deviations from the consensus in Rv0386.

detection limit (Table 1) Rv0386(1)175) had a

substan-tial GC activity of 1.0 nmol cGMPÆmg)1Æmin)1, i.e

20% of the AC activity This is unusual because all

canonical class III ACs investigated to date possess a

stringent ATP specificity [23] On the other hand it is

reminiscent of the noncanonical AC Rv1900c which

also possesses significant GC side-activity [14] The

temperature optimum of Rv0386(1)175) was 30
C, the

activation energy 76 ± 3 kJÆmol)1 (SEM, n¼ 2) and

the pH optimum was at pH 7.5–8.0 Vmax was

7.5 ± 0.8 nmol cAMPÆmg)1Æmin)1 (SEM, n¼ 4) and

the apparent Km for ATP was 0.6 ± 0.2 mm A Hill

coefficient of 1.0 ± 0.1 indicated no cooperativity for

ATP with respect to the predicted two catalytic

cen-tres The Vmax was at the lower end of bacterial class

III ACs which may reflect an unstimulated state of the

isolated CHD (Discussion) For GC activity Vmax was

2.2 ± 0.1 nmol cGMPÆmg)1Æmin)1 (n¼ 3) and the

apparent Kmfor GTP was 0.5 ± 0.03 mm with a Hill

coefficient of 1.0 ± 0.1 Thus Rv0386(1)175) had a lower turnover and a slightly higher substrate affinity

to GTP compared to ATP

Mutational analysis of Rv0386(1)175) What, if any, are the functions of those two putative substrate-binding amino acids, glutamine and aspara-gine which take the position of the canonical lysine-as-partate pair? First we removed the amide side-chains creating Rv0386(1)175)Q57A and Rv0386(1)175)N106A

to determine whether the two residues actually are necessary for catalysis Both mutants were expressed

as soluble proteins and purified to homogeneity (Fig 2) They were essentially inactive This strongly implicated that Gln57 and Asn106 interact with the substrate Next we asked whether the canonical lysine-aspartate pair would operate in Rv0386 The mutants Rv0386(1)175)Q57K, Rv0386(1)175)N106D and the double mutant Rv0386(1)175)Q57K⁄ N106D were generated, expressed and purified Rv0386(1)175)N106D was inactive Rv0386(1)175)Q57K had an AC activity

of less than 5% of wild-type activity (Table 1)

GC activity was below the detection limit (Table 1) Similarly, the purified double mutant protein Rv0386(1)175)Q57K⁄ N106D retained some AC activity (Table 1) while the GC side-activity was undetectable Thus implementation of the canonical lysine-aspartate ensemble actually was incompatible with catalytic activity This highlighted the importance of the gluta-mine-asparagine pair for substrate binding The low activity of the mutants precluded a meaningful kinetic analysis At this point, one may ask, whether Gln57 and Asn106 are indeed participating in substrate-binding or whether they are crucial for maintaining the fold of the protein In the latter case the mutants would have been inactive due to misfolding However,

we regard this as unlikely, because all mutants were soluble, purified and stable in the absence of protease inhibitors Actually, none of the previously reported mutants of the canonical lysine-aspartate couple in mammalian and mycobacterial ACs appeared to be misfolded [8,17,24]

The inactivity of Rv0386(1)175)N106D was partic-ularly remarkable, because asparagine can act as both,

a hydrogen-bond donor via its amide group and as a hydrogen-bond acceptor via its carbonyl oxygen atom

In contrast aspartate can only serve as a hydrogen-bond acceptor Therefore, we reasoned that Rv0386 uses a novel substrate-defining and -binding mechan-ism which requires a precisely positioned hydrogen-bond donor at the position of the canonical aspartate

To test this hypothesis we replaced Asn106 by a serine

R v0

6 (1

75 )

W T

Q 57 K

Q 57 A

N 10 6D

N 10 6A

Q 57

K /N 6D

N 10

6S

a

D

k

5

5

5

8

4

Q 57 E

Fig 2 Purification of recombinant proteins SDS ⁄ PAGE analysis of

purified wild-type and mutant Rv0386(1)175), 1–3 lg per lane,

visual-ized by Coomassie stain Some point mutants display a slightly

altered electrophoretic mobility.

Table 1 Activities of Rv0386(1)175)and mutants Assays were

con-ducted with 850 l M substrate and 5 m M MnCl 2 at pH 7.5 and

30
C Standard errors of the mean are included (number of

experi-ments in brackets) AC and GC activities of the mutants

Rv0386(1)175)Q57A, Rv0386(1)175)N106A and Rv0386(1)175)N106D

were below the detection limits of 0.1 and 0.2 nmolÆmg)1Æmin)1,

respectively ND, not detectable.

Enzyme

Adenylyl cyclase (nmol cAMPÆ

mg)1Æmin)1)

Guanylyl cyclase (nmol cGMPÆ

mg)1Æmin)1) Rv0386(1)175) 5.0 ± 0.6 (10) 1.0 ± 0.2 (10)

Rv0386 (1 )175)Q57K 0.2 ± 0.05 (4) ND

Rv0386(1)175)N106S 1.8 ± 0.1 (4) ND

Rv0386(1)175)Q57K ⁄

N106D

0.2 ± 0.06 (4) ND

Rv0386(1)175)Q57E 0.1 ± 0.05 (4) ND

as a potential hydrogen-bond donor and constructed

Rv0386(1)175)N106S (Fig 2) The AC activity of

purified Rv0386(1)175)N106S was 1.8 nmol cAMPÆ

mg)1Æmin)1, i.e 36% of wild-type AC activity, whereas

GC-activity was below the detection limit (Table 1)

Thus serine was not only compatible with AC

cata-lysis, but actually shifted substrate discrimination in

favour of ATP The kinetic analysis yielded a Vmax of

2.4 ± 0.4 nmol cAMPÆmg)1Æmin)1 (SEM, n¼ 4) and

a Kmof 0.4 ± 0.05 mm ATP with a Hill coefficient of

1.1 ± 0.1 Obviously, with the change from asparagine

to serine ATP-binding affinity was retained while

cata-lytic efficiency was attenuated

In analogy to the Rv0386(1)175)N106D mutant we

also generated Rv0386(1)175)Q57E On the one hand

the mutation eliminated the hydrogen-bond donor

property of the resident Q57; on the other hand a

glu-tamate at this position is highly conserved in GCs

where it may hydrogen-bond to the N1 amide and

2-amino groups of the guanine moiety [11,24] Purified

Rv0386(1)175)Q57E displayed less than 5% of the AC

activity of wild-type (Table 1) and no detectable GC

activity This confirmed that cyclase activity of Rv0386

relies specifically on the Gln57⁄ Asn106 couple

These unexpected findings cannot possibly be

recon-ciled with and discussed on the basis of the available

structural data of canonical mammalian class IIIa and

mycobacterial class IIIc catalytic domains [5,7,9,25]

nor do they parallel the findings on the noncanonical

class IIIc AC Rv1900c [14] Another novel

substrate-specifying mechanism must exist in the CHD of

Rv0386 probably brought about by peculiar structural

elements yet to be recognized

Enzymatic activity of the Rv0386 holoenzyme

To reveal a possible regulatory role of the C-terminal

putative transcription factor domain we expressed the

Rv0386 holoenzyme in E coli The majority of the

expression product ended up in inclusion bodies Yet

it was possible to solubilize a few micrograms of

enzyme with 2% CHAPS as a detergent (Fig 3)

Purification of the holoenzyme was impossible,

because it was rapidly degraded upon incubation with

the metal-affinity resin, a process which we were

unable to stop in spite of the addition of an

assort-ment of protease inhibitors (data not shown) The

specific activity of the holoenzyme was estimated to

be 3 nmol cAMPÆmg)1Æmin)1 based on comparative

protein quantification of the western blot signal

indi-cating that in the absence of effector signals the

C-terminal domains had no noticeable intrinsic

regu-latory input on the catalyst

Discussion

We characterized the unorthodox class IIIc AC Rv0386 from M tuberculosis AC activity of Rv0386 was surprising because the canonical amino acids which define substrate specificity are replaced in a non-conservative manner, glutamine-asparagine instead of lysine-aspartate All mammalian membrane-bound ACs possess a strictly conserved and spaced hexad of catalytic residues Emerging from mostly bacterial gen-ome sequencing projects deviations from this rule occur in a large number of putative AC genes Actu-ally, predicted open reading frames for ACs exist where all six canonical amino acids are replaced non-conservatively [4]

Viewed from the structures of mammalian ACs those predicted proteins do not look like they could possibly have any AC activity unless alternate mecha-nisms of catalysis or substrate-binding exist for the conversion of ATP to cAMP [4] However, the first structures of a variant AC were recently obtained with Rv1900c [14] There the histidine residue which substi-tutes the canonical transition-state stabilizing aspara-gine does not contact the substrate and mutagenesis shows that it appears not to be involved in catalysis Furthermore the asparagine-aspartate couple which replaces the usual substrate-specifying lysine-aspartate pair does not bind to the purine moiety and is dispen-sable for catalysis This implies that the preference of Rv1900c for ATP over GTP is governed by other determinants, e.g general steric constraints of the purine-binding pocket

a D k 6 1

5 5

5

Fig 3 Rv0386 holoenzyme Solubilized Rv0386 holoenzyme (calcu-lated molecular mass, 118 kDa) was analyzed by western blot with

a commercial anti-RGSH 4 Ig The signal of the holoenzyme corres-ponds to 80 ng protein.

In contrast to Rv1900c, AC Rv0386 shows a

differ-ent mechanism, because the glutamine-asparagine

couple of Rv0386 is specifically needed for catalysis

Removal of either amide side-chain as in Q57A or

N106A mutants abrogated cyclase activity Thus

mul-tiple variants of catalytic pockets seem indeed to exist

in class III ACs The inability of Rv0386 to operate

with a consensus lysine-aspartate pair, as demonstrated

by the catalytic incompetence of the Q57K, N106D,

and Q57K⁄ N106D mutants, suggests that Gln57 ⁄

Asn106 do bind the purine moiety of the substrate,

but in a different mode compared to canonical ACs

The high GC side-activity of Rv0386 indicates that

both, adenine and guanine can be accommodated in

the substrate-binding pocket via Gln57⁄ Asn106 How

can the purine be bound by the two amide

side-chains? In all structures of canonical ACs, i.e

mam-malian AC, trypanosomal AC and mycobacterial AC

Rv1264 the lysine-aspartate couple forms a salt bridge

[5,7,26] Even in Rv1900c the asparagine-aspartate

pair is connected by a hydrogen bond when the

sub-strate-binding pocket is unoccupied [14] It is

there-fore plausible to assume that Gln57 and Asn106 are

similarly bonded in Rv0386 We propose that Gln57

and Asn106 are arranged in positions that could

accommodate either a guanine or an adenine moiety

(Fig 4A,B) Mutation of either one would therefore

be expected to abolish all cyclase activity, as has been

observed experimentally The results with the N106S

point mutation, i.e maintaining cyclase activity and

enhancing ATP substrate specificity, are compatible

with the proposed mechanism, because Ser106 could

pair with Gln57 in the ATP-binding conformation

(Fig 4C) It should be noted that a related ‘amide

switch’ mechanism of purine binding and specificity

has previously been identified in mammalian cyclic-nucleotide phosphodieserases based on crystal struc-tures [27–29]

The specific activity of Rv0386 was robust and easily measurable with precision, yet, it represents a low activity CHD when compared to other bacterial class III ACs Nevertheless this does not mean that cAMP production by Rv0386 is physiologically irrelevant Both, high activity and low activity CHDs have been described previously in M tuberculosis High activity CHDs are Rv1625c (2 lmol cAMPÆmg)1Æmin)1) [8], Rv1264 (1 lmol cAMPÆmg)1Æmin)1) [17], Rv1900c (1 lmol cAMPÆmg)1Æmin)1) and Rv1647 (3 lmol cAMPÆ

mg)1Æmin)1) [19] Low activity CHDs are present

in Rv1318c (0.3 nmol cAMPÆmg)1Æmin)1), Rv1319c (7 nmol cAMPÆmg)1Æmin)1), Rv1320c (0.2 nmol cAMPÆ

mg)1Æmin)1) and Rv3645 (9 nmol cAMPÆmg)1Æmin)1) [18] The low activity CHD of Rv3645 can be stimula-ted by almost two orders of maginitude via the adjoin-ing HAMP domain [18] Thus a regulatory input can greatly enhance catalytic efficiency of a low activity CHD in the background of a holoenzyme We envisage that the Rv0386 holoenzyme, which has a low AC activity comparable to the isolated CHD, will be stimu-lated by an as yet unknown effector A further argu-ment in favour of a physiological relevance of the AC activity of Rv0386 lies in the evolution of the protein The glutamine-asparagine couple is not a degenerate mutation, but specifically required for catalysis Thus

AC activity appears to have been retained by a pressure

of selection

However, the biological function of Rv0386 in

M tuberculosis is unclear at this point Recently, in a transposon-mutagenesis screen of M tuberculosis a knock out mutant of Rv0386 (or all other presumed

A R v 3 6 - A T P B R 0 8 6 - G T P

6 1 n s A

7 n l G

P P P b i R

N N

N

N

N H

N H H

O N H

H

6 1 n s A

7 n l G

P P P b i R

N N

N

N O

H2 H

N

O

H H N

O H

H

C R v 3 6 - N 1 6 S - A T P

6 1 r e S

7 n l G

P P P b i R

N N

N

N

N H

N H H

O H

Fig 4 Proposed mode of purine binding Adenine and guanine binding in Rv0386 by paired Gln57 and Asn106 residues is based on a pos-sible amide switch (compare A and B) (C) Increased specificity for ATP in the Rv0386 (1 )175)N106S mutant are the consequence of the

hydrogen bond donor property of the serine The ribose-5¢-triphosphate moiety is abbreviated by rib-P-P-P.

mycobacterial AC genes) was viable under cell culture

conditions [30] Considering the elusive pathogenic

pathways of mycobacteria in its host this does not

exclude a vital function of Rv0386 under

pathophysio-logical survival conditions This suggestion is

partic-ularly justified because a comparative genomic analysis

of several mycobacterial strains identified Rv0386 as

one of a few genes which are specifically retained in

the M tuberculosis complex while being lost in other

strains, e.g it is absent in M smegmatis [31]

The substrate specificity of Rv0386 may be

deter-mined by the concentrations of available ATP and

GTP at the cellular location of the enzyme, yet the

intracellular concentrations of ATP and GTP in

M tuberculosis are unknown to date In fact, with the

exception of Synechocystis [32] meaningful and

unequi-vocal cellular cGMP levels have not been reported to

date in M tuberculosis nor in any other bacteria In

the finished genome of M tuberculosis 10 putative

pro-teins were identified which contain a cyclic

nucleotide-binding domain [15] However, no cGMP specificity

has been predicted for any of these proteins We are

aware, of course, that this does not exclude the

exist-ence of novel, hitherto unknown cGMP binding

pro-teins in the pathogen

In conclusion the characterization of AC Rv0386 in

this study reveals novel aspects in several respects It

has a completely novel mechanism of substrate binding

so far not observed in other class III ACs It has a

rather striking new domain composition comprising an

AAA-ATPase and transcription factor module with

broad physiological implications to be elucidated

Experimental procedures

Materials

Radiolabelled nucleotides were from Hartmann Analytik

(Braunschweig, Germany) Genomic DNA from M

tuber-culosiswas from Dr Boettger (University of Zu¨rich Medical

School, Switzerland) pBluescriptII SK(–) (Stratagene,

Hei-delberg, Germany) was used for general cloning and pQE30

(Qiagen, Hilden, Germany) for expression in Ni2+

-nitrilo-triacetic acid-agarose slurry was from Qiagen The

anti-RGSH4antibody was obtained from Qiagen, the secondary

antibody from Dianova, (Hamburg, Germany) Peroxidase

detection was carried out with the ECL-Plus kit

(Amer-sham-Life Sciences)

Plasmid Construction

The open reading frame of gene Rv0386 (GenBank

Acces-sion Number BX842573) was amplified by PCR using

specific primers and genomic DNA as a template and a BamHI and a HindIII site were added to the 5¢- and 3¢-ends, respectively To remove the internal BamHI site a silent AfiT mutation was introduced at nucleotide 57 The PCR product was inserted into pQE30, adding an N-terminal MRGSH6GS tag Similarly, the catalytic domain (Rv0386(1)175)) was fitted with a 5¢ BamHI and a 3¢ HindIII site and inserted into pQE30 Point mutations were introduced by PCR using the expression cassette as a template and standard molecular biology techniques The correctness of all DNA inserts was checked by double-stranded DNA sequencing Primer sequences are available

on request

Expression and purification of proteins Plasmids containing Rv0386(1)175) or its mutants were transformed into E coli BL21(DE3)[pRep4] Protein expression was induced by 60 lm isopropyl-thio-b-D-gal-actoside for 3–5 h at 22
C Bacteria were washed once with buffer (50 mm Tris⁄ HCl, 1 mm EDTA, pH 8) and stored at)80
C For purification cells from 200 to 600 mL culture were suspended in 25 mL of lysis buffer (50 mm Tris⁄ HCl, pH 8, 50 mm NaCl, 10 mm 2-mercaptoethanol), lysed by sonication for 30 s and treated for 30 min with 0.2 mgÆmL)1 lysozyme on ice Subsequently 5 mm MgCl2 and 20 lgÆmL)1DNAseI were added for 30 min After

cen-trifugation (31 000 g, 30 min) 15 mm imidazole pH 8 and

250 mm NaCl (final concentrations) were added to the supernatant Protein was equilibrated for a minimum of

60 min with 250 lL Ni2+⁄ nitrilotriacetic acid agarose on ice, then transferred to a column and successively washed with 3 mL each of buffer A (lysis buffer containing 5 mm imidazole, 400 mm NaCl and 2 mm MgCl2), buffer B (lysis buffer containing 15 mm imidazole, 400 mm NaCl and

2 mm MgCl2) and buffer C (lysis buffer containing 15 mm imidazole, 10 mm NaCl and 2 mm MgCl2) The protein was eluted with 0.4 mL of buffer D (lysis buffer containing

150 mm imidazole, 10 mm NaCl and 2 mm MgCl2) Puri-fied proteins were dialyzed against buffer E (50 mm Tris⁄ HCl, pH 8, 10 mm NaCl, 2 mm 2-mercaptoethanol, 20% glycerol) and stored at )20
C The enzyme was stabile for several weeks at least

Cyclase assays

AC activity was determined at 30
C for 20 min in 100 lL [33] The reactions contained 50 mm 3-(N-morpholino)-propanesulfonic acid pH 7.5, 22% glycerol, 5 mm MnCl2,

850 lm [32P]ATP[aP] and 2 mm [2,8-3H]cAMP The kinetic analysis was conducted from 10 lm to 2.3 mm ATP and kinetic constants were derived from a Hanes-Woolfe plot

GC activity was determined identically by using guanine nucleotides instead of the respective adenine nucleotides [34]

Western blot analysis

Protein was mixed with sample buffer and subjected to

SDS⁄ PAGE (15%) The gel was blotted onto

poly(vinylid-ene difluoride) membranes and probed sequentially with a

commercial anti-RGSH4 Ig and with a 1 : 5000 dilution of

peroxidase conjugated goat anti-(mouse IgG) Ig as a

secon-dary antibody

Sequence analyses

INTERPRO-scans (http://www.ebi.ac.uk/InterProScan/index

html) and smart analysis (simple modular architecture

research tool; http://smart.embl-heidelberg.de/) were

per-formed

Acknowledgements

This work was supported by the Deutsche

Forschungs-gemeinschaft

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