Discovery of posttranslational maturation byself-subunit swappingZhemin Zhou*†, Yoshiteru Hashimoto*†, Kentaro Shiraki‡, and Michihiko Kobayashi*§

*Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, and ‡Institute of Applied Physics, University of Tsukuba, 1-1-1Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved August 7, 2008 (received for review April 9, 2008)

Several general mechanisms of metallocenter biosynthesis havebeen reported and reviewed, and in all cases, the components orsubunits of an apoprotein remain in the final holoprotein. Here, wefirst discovered that one subunit of an apoenzyme did not remainin the functional holoenzyme. The cobalt-containing low-molecu-lar-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrousJ1 consists of �- and �-subunits encoded by the nhlBA genes,respectively. An ORF, nhlE, just downstream of nhlBA, was foundto be necessary for L-NHase activation. In contrast to the cobalt-containing L-NHase (holo-L-NHase containing Cys-SO2

� and Cys-SO� metal ligands) derived from nhlBAE, the gene products de-rived from nhlBA were cobalt-free L-NHase (apo-L-NHase lackingoxidized cysteine residues). We discovered an L-NHase maturationmediator, NhlAE, consisting of NhlE and the cobalt- and oxidizedcysteine-containing �-subunit of L-NHase. The incorporation ofcobalt into L-NHase was shown to depend on the exchange of thenonmodified cobalt-free �-subunit of apo-L-NHase with the cobalt-containing cysteine-modified �-subunit of NhlAE. This is a post-translational maturation process different from general mecha-nisms of metallocenter biosynthesis known so far: the unexpectedbehavior of a protein in a protein complex, which we named‘‘self-subunit swapping.’’

metalloenzyme � modification � sulfinic acid � sulfenic acid � chaperone

In all living cells, proteins and protein complexes often changechemically or structurally before becoming functional. Alter-

ations like modifications or binding of a metal or ligand areobserved at a specific amino acid or in a specific region ofproteins (1, 2). Metalloproteins have been characterized inten-sively for decades, yet investigators only recently have focused onthe mechanisms of biological metallocenter assembly (1, 3). Nitrilehydratase (NHase; EC4.2.1.84) (4, 5), which is composed of �- and�-subunits, contains either a nonheme iron (6–9) or noncorrincobalt ion (10–12) in a ligand environment that includes twooxidized cysteine residues [CXLC(SO2H)SC(SOH)] (8, 9, 13, 14).The enzyme catalyzes the hydration of a nitrile to the correspondingamide, followed by consecutive reactions: amide 3 acid 3 acyl-CoA by amidase (15) and acyl-CoA synthetase (16, 17), respec-tively. Rhodococcus rhodochrous J1 produces high- and low-molecular-mass NHases (H-NHase and L-NHase), which exhibitdifferent physicochemical properties and substrate specificities (4).In both H- and L-NHases, cobalt acts as an active center for theproduction of acrylamide and nicotinamide. Acrylamide is manu-factured at the industrial level not only in Japan but also in theUnited States and France (18, 19).

Ferric-NHases require activators for functional expression inRhodococcus sp. N-771 (20), Pseudomonas chlororaphis B23 (21),Pseudomonas putida 5B (22), and Rhodococcus sp. N-774 (23). Aproposed metal-binding motif, CXCC, in the NHase activator ofRhodococcus sp. N-771 has been identified, and the activators forFe-type NHases have been shown to act as metallochaperones(24). Although there are some ORFs including regulatory genesin the H- and L-NHase gene clusters (25, 26), only the gene ofa cobalt transporter (NhlF), which mediates the cobalt transportinto the cell, has been characterized (27). However, the mech-

anism for the incorporation of cobalt into Co-NHases remainsunclear. Here, our results reveal that the incorporation of acobalt ion into L-NHase involves a thus-far unknown posttrans-lational mechanism of metallocenter biosynthesis.

ResultsNecessity of nhlE for L-NHase Activation. To express L-NHase, weused a host–vector system for an actinomycete, Rhodococcusfascians DSM43985 (used as the host for plasmid pREIT19),which was recently developed in our laboratory (Y.H., T.Ishikawa, Z.Z., H. Maseda, H. Higashibata, and M.K., unpub-lished work). Plasmid pREIT-nhlBA (Fig. 1A), comprising thenhlBA genes (encoding the �- and �-subunits of L-NHase) inpREIT19, was constructed and used to express L-NHase. Thebands of the �- and �-subunits of L-NHase were obvious onSDS/PAGE (Fig. 1B), whereas the activity in cell-free extracts ofthe Rhodococcus transformant was very low (0.21 � 0.01 units/mg). On the contrary, R. rhodochrous ATCC12674 harboringpLJK60 (Fig. 1 A), which contains nhlBAE [i.e., the nhlBA andnhlE (an ORF just downstream of nhlBA) genes], showed highL-NHase activity (10), suggesting that the nhlE gene, which is onthe same transcription unit as nhlBA, is necessary for functionalexpression of L-NHase. Then, plasmid pREIT-nhlBAE (Fig. 1 A)carrying the nhlBAE genes was constructed and used for L-NHase expression. As shown in Fig. 1B, the amount of L-NHaseexpressed was similar to that of pREIT-nhlBA, and the L-NHaseactivity (8.62 � 0.40 units/mg) in cell-free extracts was muchhigher than that of pREIT-nhlBA. This finding revealed thatnhlE was responsible for L-NHase activation.

Differences Between the L-NHases Derived from nhlBA and nhlBAE. Todetermine the differences between the L-NHases derived fromnhlBA and nhlBAE, we completely purified and characterizedthe two enzymes. As a result, L-NHase derived from nhlBAE wasfound to be a functional tetramer (�2�2), exhibiting specificactivity of 321 � 6 units/mg [Fig. 1 C and D, Table 1, supportinginformation (SI) Fig. S1, and Discussion in SI Text]. A het-erodimer (��) and a heterotetramer (�2�2) of L-NHase exhib-iting low activity were isolated when nhlBA was expressed (Fig.1 C and D, Table 1, Fig. S2, and Discussion in SI Text). The cobaltcontent [which is essential for the activity (4, 28)] of L-NHasederived from nhlBAE was found to be 0.88 � 0.04 mol/mol of ��,whereas those of the heterodimer and heterotetramer derivedfrom nhlBA were very low (Table 1). Whereas the active enzyme is

Author contributions: M.K. designed research; Z.Z. and Y.H. performed research; Z.Z. andY.H. contributed new reagents/analytic tools; Z.Z., Y.H., K.S., and M.K. analyzed data; andZ.Z., Y.H., and M.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

†Z.Z. and Y.H. contributed equally to this work.

§To whom correspondence should be addressed. E-mail: sec-mmb@agbi.tsukuba.ac.jp. Fax:�81-29-853-4605.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803428105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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presumed to contain the two oxidized cysteine ligands (8, 9, 13, 14),the apoprotein is likely to be nonmodified, judging from previousstudies of NHase (12) and the related enzyme thiocyanate hydro-lase (SCNase) (29). These findings suggest that the gene productsderived from nhlBA are apo-L-NHases (apo-�� and apo-�2�2,respectively), in contrast to the holo-L-NHase (holo-�2�2) derivedfrom nhlBAE.

Isolation of a Cobalt-Containing Mediator, NhlAE. To clarify the roleof nhlE in the activation of L-NHase, we at first purified the geneproduct (NhlE) from the transformant harboring pREIT-nhlBAE. As a result, NhlE was confirmed to form a complex (i.e.,NhlAE) with the �-subunit of L-NHase (Fig. 1C). The molecularmasses determined on gel filtration and sedimentation equilib-rium analyses indicated that the complex consists of a hetero-trimer, �e2 (NhlE is denoted as e) (Fig. 1D, Fig. S3, andDiscussion in SI Text). The purified �e2 exhibited no L-NHaseactivity, whereas it contained a cobalt ion (0.87 � 0.03 mol/molof �e2) (Table 1). Hereafter, the cobalt-containing �e2 is re-ferred to as holo-�e2. Next, we tried to determine whether or notNhlE could be expressed alone. When plasmid pREIT-nhlE(Fig. 1 A) was constructed and transformed in R. fascians

DSM43985, no expression was observed. Furthermore, NhlEwas not expressed in the transformant harboring pREIT-nhl�AE(Fig. 1 A), in which the N-terminal 24 residues of the �-subunithad been deleted to yield a truncated �-subunit with Met-25 asthe first residue. Holo-�e2 was produced successfully in thetransformant harboring pREIT-nhlAE (Fig. 1 A and B and Table1). These findings demonstrate that NhlE could not be translatedwithout the nhlA gene or that translated NhlE could be degradedrapidly by protease in the absence of the �-subunit of L-NHase.We successfully isolated the �-subunit and NhlE from holo-�e2through protein denaturation and renaturation (Fig. S4), andfound that the resultant purified �-subunit contained cobalt(0.80 � 0.02 mol/mol of �), whereas the resultant NhlE did not.

Conversion of Apo-L-NHase to Holo-L-NHase by Holo-�e2. To eluci-date the role of NhlAE in the formation of active L-NHase, wemixed apo-�� or apo-�2�2 (final, 0.1 mg/ml) with the purifiedholo-�e2 (final, 0.2 mg/ml) and then incubated them in 10 mMpotassium phosphate buffer (KPB) (pH 7.5) at 28°C. Surpris-ingly, we found that the L-NHase activity in the mixtures becamehigher as the incubation time increased, the highest activity beingobserved after 12 h (Fig. S5). As the holo-�e2 concentrationincreased, the activation rate became faster, and the highestactivity was obtained within 4 h in the case of 0.8 mg/ml holo-�e2(Fig. S5). Although there is a possibility that the apo-L-NHaseis not a required intermediate in the maturation process in vivo,this result suggests that the apo-L-NHase could be posttransla-tionally activated by holo-�e2 in vitro. Although no significantchanges were observed in the gel-filtration profiles of themixture of apo-�2�2 and a 2-fold excess of holo-�e2 at 0 h andafter 12 h (Fig. 2A), the mixture of apo-�� and �e2 after 12 h gavea new peak corresponding to a heterotetramer, �2�2 (Fig. 2B).From the mixtures, we then purified each resultant L-NHasederived from apo-�2�2 (R-�2�2) or apo-�� (R-��) (Fig. 2 C andD) and found that its enzyme activity (328 � 10 and 326 � 8units/mg, respectively) and cobalt content (0.95 � 0.03 and0.96 � 0.04 mol/mol of ��, respectively) were similar to those ofholo-�2�2 and that R-�� changed into the heterotetramer, �2�2(Table 1). The cobalt content of the �e2 purified from themixture was remarkably decreased; the cobalt ion was stoichio-metrically incorporated from holo-�e2 into each apo-L-NHase(Discussion in SI Text). Through far-UV CD and UV-Visabsorption spectra analyses, we carried out a detailed compar-ison of each property between apo-��, apo-�2�2, R-��, R-�2�2,and holo-�2�2. The spectra of both apo-�� and apo-�2�2 aredifferent from those of holo-�2�2 (Fig. S6), indicating that thesecondary structure of apo-�� or apo-�2�2 is not the same as thatof holo-�2�2. On the contrary, the CD and UV-vis spectra ofR-�� and R-�2�2 are similar to those of holo-�2�2 (Fig. S6).Holo-�2�2, R-��, and R-�2�2 all exhibited an extra shoulder inthe 300- to 350-nm region. It has been reported that theabsorption in the 300- to 350-nm region of Co-NHase reflectsS 3 Co3� charge transfer (11, 30), because synthetic low-spinCo3�–thiolate complexes give ligand-to-metal charge transferbands at 280 nm (31–33). These results suggested that theCo–ligand environment of R-�� or R-�2�2 is the same as that ofholo-�2�2. Together with the inclusion of cobalt in L-NHase,these phenomena thus demonstrate that �e2 participates in theposttranslational maturation of L-NHase.

�-Subunit Exchange Between Apo-L-NHase and Holo-�e2 During Post-translational Maturation of L-NHase. Our findings could be ex-plained by two possible mechanisms: One is that only cobaltions are transferred from holo-�e2 to the apo-�-subunit ofapo-L-NHases, and the other is that the apo-�-subunit inapo-L-NHases is replaced by a holo-�-subunit in holo-�e2.Subsequently, site-directed mutagenesis and N-terminal aminoacid sequence analysis were carried out to confirm the source of

Fig. 1. Expression and purification of L-NHase and holo-�e2. (A) Geneticorganization for the construction of a set of plasmids. The asterisk indicatesthe mutant nhlA and nhlB genes. (B) SDS/PAGE of a cell-free extract of each R.fascians DSM43985 transformant. (C and D) SDS/PAGE (C) and gel filtrationprofiles (D) of purified L-NHases and NhlAE. The corresponding genes on theexpression plasmid (pREIT series) used for the preparation of L-NHase andNhlAE are shown in italics after each enzyme name.

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each �-subunit in the resultant L-NHases derived from apo-L-NHases. We designed a mutant gene [nhlBA-(�-A3G)] in whichAla-3 in the �-subunit was substituted by Gly, constructingpREIT-nhlBA-(�-A3G) (Fig. 1 A) and then purified two mu-tants, apo-(�-A3G)� and apo-(�-A3G)2�2. No significant dif-ferences in the specific activities of the purified enzymes wereobserved between apo-�� and apo-(�-A3G)� or between apo-�2�2 and apo-(�-A3G)2�2 (Table 1). We mixed each of apo-(�-A3G)� and apo-(�-A3G)2�2 with holo-�e2, and confirmed theposttranslational activation of L-NHase. On N-terminal aminoacid sequence analysis of the �-subunits in the resultant purifiedL-NHases [R-(�-A3G)� and R-(�-A3G)2�2], position 3 of eachof �-subunit was shown to be Ala (Table 1), demonstrating thatthe apo-�-subunit of each apo-L-NHase is replaced by theholo-�-subunit of holo-�e2.

Investigation of Cysteine Modification in Holo-�e2 and Cobalt-Free�e2 (Apo-�e2). Metal ion binding is responsible for the posttrans-lational modification of the two cysteines in the �-subunit inNHase (1, 12), but such modifications cannot be observed inapo-NHase or cobalt-free SCNase (12, 29). These results permitus to speculate that the cysteine residues are modified inholo-�e2, but not in apo-�e2 (purified from the culture in theabsence of cobalt) (Table 1). We analyzed holo-�e2 and apo-�e2by MALDI-TOF MS. The holo-�-subunit isolated from holo-�e2 and the apo-�-subunit isolated from apo-�e2 were treatedwith trypsin after reduction and carboxamidomethylation. The

molecular mass of the tryptic peptide containing all metal ligandresidues, E83MGVGGMQGEEMVVLENTGTVHNMV-VC109TLC112SC114YPWPVLGLPPNWYK128 (EK46), was mea-sured. As a result, Cys-SO2H was detected in the holo-�-subunitbut not in the apo-�-subunit (Fig. 3 and Discussion in SI Text).Although the occurrence of Cys-114-SOH modification has notbeen confirmed because of its chemical instability (34), theseresults strongly suggest that the oxidized cysteine residues (�Cys-112-SO2H and �Cys-114-SOH) would exist in holo-�e2, but notin apo-�e2. Considering that the holo-�-subunit of holo-L-NHase is derived from holo-�e2 through �-subunit exchange, thecorresponding two cysteine residues in holo-L-NHase would bemodified.

Driving Force for �-Subunit Exchange Between Apo-L-NHase andHolo-�e2. Posttranslationally modified Cys-SO2H and Cys-SOHhave deprotonated Cys-SO2

� and Cys-SO� structures, respec-tively (7), and the deprotonated Cys-SO2

� and Cys-SO� in theholo-�-subunit form salt bridges with two arginines of the�-subunit (which are conserved in all known Co-type andFe-type NHases) in the holo-enzyme (8, 9, 13, 14). The salt-bridge mechanism postulates that the electrostatic networkaround a cobalt ion including the two negative modified cys-teines (Cys-112 and Cys-114) in the �-subunit and the twoarginines (R52 and R157) in the �-subunit play a key role instabilizing the subunit interface (Fig. 4A). There is a possibilitythat the electrostatic force needed to form the salt bridges

Table 1. Characterization of the purified L-NHases, NhlAEs, and resultant (R-) L-NHases derived on mixing with holo-�e2

Protein

L-NHaseactivity,units/mg Structure

Co content(mol of ions/mol

of protein)

N-terminalsequence of

�-subunit

holo-�2�2 [nhlBAE] 321 � 6 �2�2 0.88 � 0.04/�� N.T.apo-�2�2 [nhlBA] 38.2 � 1.1 �2�2 0.036 � 0.001/

��

N.T.

apo-�� [nhlBA] 4.13 � 0.16 �� 0.030 � 0.001/��

N.T.

holo-�e2 [nhlBAE] 0 �e2 0.87 � 0.03/�e22TAHNP6

holo-�e2 [nhlAE] 0 �e2 0.86 � 0.04/�e22TAHNP6

R-�2�2 328 � 10 �2�2 0.95 � 0.03/�� N.T.R-�� 326 � 8 �2�2 0.96 � 0.04/�� N.T.apo-(�-A3G)2�2 [nhlBA-(�-A3G)] 36.3 � 1.7 �2�2 0.032 � 0.001/

��

2TGHNP6

apo-(�-A3G)� [nhlBA-(�-A3G)] 3.26 � 0.12 �� 0.034 � 0.001/��

2TGHNP6

R-(�-A3G)2�2 331 � 12 �2�2 N.T. 2TAHNP6

R-(�-A3G)� 318 � 9 �2�2 N.T. 2TAHNP6

apo-�e2 [nhlAE] 0 �e2 0.025 � 0.001/�e2

N.T.

R-(�-A3G)2�2* 38.6 � 1.3 �2�2 N.T. 2TGHNP6

R-(�-A3G)�* 4.36 � 0.10 �� N.T. 2TGHNP6

apo-(�-A3G)2(�-R52A/R157A)2

[nhlBA-(�-A3G)(�-R52A/R157A)]0 �2�2 0.029 � 0.001/

��

2TGHNP6

apo-(�-A3G)(�-R52A/R157A)[nhlBA-(�-A3G)(�-R52A/R157A)]

0 �� 0.031 � 0.001/��

2TGHNP6

R-(�-A3G)2(�-R52A/R157A)2 0 �2�2 N.T. 2TGHNP6

R-(�-A3G)(�-R52A/R157A) 0 �� N.T. 2TGHNP6

apo-�2(�-R52A/R157A)2

[nhlBAE-(�-R52A/R157A)]0 �2�2 0.045 � 0.001/

��

N.T.

apo-�(�-R52A/R157A)[nhlBAE-(�-R52A/R157A)]

0 �� 0.040 � 0.001/��

N.T.

holo-�e2 [nhlBAE-(�-R52A/R157A)] 0 �e2 0.85 � 0.02/�e2 N.T.

The corresponding genes on the expression plasmid (pREIT series) used for the preparation of L-NHase and NhlAE are shown inparentheses after each protein. The third N-terminal amino acid of each �-subunit is shown in bold. The values represent the means �SD for at least triplicate independent experiments. N.T., not tested.*R-(�-A3G)2�2 derived on mixing with apo-�e2; R-(�-A3G)� derived on mixing with apo-�e2.

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between the two negatively charged modified cysteines of the�-subunit in holo-�e2 and the two positively charged arginines ofthe �-subunit in apo-enzymes triggers �-subunit exchange. Thishypothesis is supported by the finding that no �-subunit ex-

change occurred between apo-�e2 and apo-L-NHases in vitrobecause of the absence of cysteine oxidation in the apo-�-subunitof apo-�e2 (Table 1 and Fig. 4B). In addition, no �-subunitexchange occurred between holo-�e2 and the apo-L-NHasemutants [(apo-(�-A3G)2(�-R52A/R157A)2 and apo-(�-A3G)(�-R52A/R157A)] derived from nhlBA-(�-A3G)(�-R52A/R157A),which has a double mutation at the two arginines that eliminates thepositive charge in the �-subunit (Table 1). Furthermore, theholo-�e2 and two types of apo-L-NHases [(apo-�2(�-R52A/R157A)2) and apo-�(�-R52A/R157A)] were purified from theRhodococcus transformant containing pREIT-nhlBAE-(�-R52A/R157A) (Table 1), indicating that elimination of the positive chargeof the two arginines in the �-subunit did not cause �-subunitswapping at all, resulting in accumulation of the holo-�e2 and twotypes of apo-L-NHases in vivo. These results suggest that theelectrostatic attraction that forms the salt bridges would play anessential role in posttranslational maturation of L-NHase in vitroand in vivo.

DiscussionHere, we discovered a thus-far unknown maturation mechanismfor a protein during posttranslational activation. In L-NHase, (i)

Fig. 2. Maturation of apo-L-NHases with holo-�e2. (A) Elution profiles ofapo-�2�2 mixed with NhlAE at 0 and 12 h upon gel filtration. (B) Elutionprofiles of apo-�� mixed with holo-�e2 at 0 and 12 h upon gel filtration. (C)SDS/PAGE of the mixture of apo-�2�2 and holo-�e2 (lanes 0 and 12 h), thepurified R-�2�2 (lane R-�2�2), and the �e2 (lane R-�e2) purified from themixture. (D) SDS/PAGE of the mixture of apo-�� and holo-�e2 (lanes 0 and12 h), the purified R-�� (lane R-��), and the �e2 (lane R-�e2) purified from themixture. In A and B, 100 �l of each sample (containing 30 �g of proteins) wasapplied.

Fig. 3. MALDI-TOF MS spectra of the metal-binding peptide, EK46, of theapo-�-subunit (A) and holo-�-subunit (B). The mass peak with an m/z value of5242.36 (A) corresponds to the [M�H]� ion of EK46 with three carboxam-idomethylated (CAM�) cysteines (calculated m/z value � 5243.11), and thatwith an m/z value of 5217.93 (B) corresponds to the [M�H]� ion of EK46 witha Cys-SO2H and two CAM-cysteines (calculated m/z value � 5218.06). SeeDiscussion in SI Text for more details.

Fig. 4. Self-subunit swapping maturation of L-NHase. (A) A model of thenoncorrin cobalt center of L-NHase. The model is based on all known crystalstructures of Co-type NHases (13, 14) and Fe-type NHases (8, 9). Atoms areshown in different colors: pink for Co, black for C, red for O, yellow for S, andblue for N. The salt-bridge networks formed between the modified cysteinesand the two arginines are shown as red dotted lines. (B) The proposed het-erodimer complexes of holo-L-NHase (holo-��) and apo-L-NHase (apo-��), andheterotrimer complexes of apo-�e2 and holo-�e2. The two cysteines in the apo-�-subunit are denoted as �SH, and the two modified cysteines in the holo-�-subunit as �SO� and SO2

�, respectively. The two arginines in the �-subunit aredenoted as �Arg�. (C) Biosynthesis of apo-L-NHases and holo-�e2 (Step 1), andself-subunit swapping maturation in vitro (Step 2). (D) Self-subunit swappingmaturation in vivo. The cobalt-containing �-subunit is shown in pink.

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cobalt-containing NhlAE consists of NhlE and the Co-boundand cysteine-oxidized �-subunit of L-NHase, (ii) the Co-boundand cysteine-oxidized �-subunit dissociates from NhlE andreplaces a non-Co-bound and non-cysteine-oxidized form of the�-subunit (that forms an apo-�� or apo-�2�2 complex) to yielda holoenzyme. Several general mechanisms of metallocenterbiosynthesis have been reported and reviewed (1). In all cases,the components of the apoprotein remain in the final holopro-tein. However, our results demonstrate that the cobalt-free andnon-cysteine-oxidized �-subunit in the apoenzyme never re-mains in the final functional protein, being eventually replacedby a different cobalt-containing cysteine-modified �-subunit,which yields a holoenzyme. This posttranslational activation isquite different from thus-far known general mechanisms ofmetallocenter biosynthesis and reveals the unexpected behaviorof a protein in a protein complex. Therefore we named it‘‘self-subunit swapping’’ maturation. Self-subunit swapping hasthe following features: (i) the swapping is not between identicalsubunits but, rather, between an unmodified subunit and amodified one with the same sequence, (ii) the apo-enzyme isaltered to the holo-enzyme only through one-time-swappingwithout any other process, and (iii) an apoprotein subunit neverremains in the holoprotein. Self-subunit swapping is a conceptcompletely different from thus-far known processes involvingswapping between an apoprotein and a chaperone or between acofactor and a chaperone, regarding the metallocenter biosyn-thesis of several proteins [e.g., superoxide dismutase (35, 36),ribonucleotide reductase (37–39), and dinitrogenase (40), re-spectively]. NhlE, exhibiting a surprising protein function, shouldbe recognized as a self-subunit swapping chaperone in contrastwith metallochaperones in metallocenter biosynthesis (36), andmolecular chaperones in protein folding (41, 42).

Although expression of the nhlBAE genes resulted in theformation of both holo-�2�2 and holo-�e2, the expression ofnhlBAE-(�-R52A/R157A) resulted in the formation of a holo-�e2 and two types of apo-L-NHases (Table 1). These findingssuggest that the biosynthesis of L-NHase in vivo involves thefollowing two steps: (i) the �-subunit, NhlE and a cobalt ion firstform a complex to become a Co-bound and cysteine-oxidizedholo-�e2, and simultaneously the �-subunit associates with the�-subunit yielding a non-Co-bound and non-cysteine-oxidizedapo-L-NHase; (ii) the �-subunit exchange between the Co-bound and cysteine-oxidized holo-�e2 and non-Co-bound andnon-cysteine-oxidized apo-L-NHases occurs through the drivingforce of self-subunit swapping to complete the L-NHase matu-ration posttranslationally. When the Rhodococcus transformantcarrying pREIT-nhlBAE-(�-R52A/R157A) is used for L-NHaseexpression, it is easily expected that holo-�e2 derived from nhlAEand apo-L-NHases derived from nhlBA-(�-R52A/R157A) areformed. Because the driving force required for self-subunitswapping could not be provided with the elimination of thepositive charge of arginines in the �-subunit, the intermediatesin the process of L-NHase activation (i.e., apo-L-NHases and

holo-�e2) accumulated in the cells. If there is a mechanism otherthan self-subunit swapping for cobalt incorporation into apo-L-NHases, apo-L-NHases should become holo-L-NHases throughthis mechanism. The accumulation of apo-L-NHases suggests thatself-subunit swapping would be the sole maturation mechanism forL-NHase and self-subunit swapping would occur in vivo. Wesummarize our findings demonstrating self-subunit swapping mat-uration of L-NHase in vitro and in vivo in Fig. 4 C and D.

In Bacillus pallidus RAPc8, a Co-NHase activator, P14K, hasbeen found to be necessary for its functional NHase expressionand proposed to act as a subunit-specific chaperone (� � P14K3 [�P14K] � Co 3 [�P14K-Co] � � 3 ��-Co � P14K) (43),but this hypothesis has never been explicitly tested. We actuallyisolated the complex (�e2), and demonstrated self-subunit swap-ping between apo-��/apo-�2�2 and �e2. Moreover, we con-firmed that the complex (the �-subunit being either Co free orCo containing) is stable even after the posttranslational activa-tion process, although the [�P14K] complex is proposed to benecessarily a transient one (43). During the purification of theholoenzyme, apoenzymes and �e2, the �-subunit, the �-subunit,and NhlE were not observed as a single protein, but theseproteins were observed as the corresponding complexes. There-fore, NhlE functions as a self-subunit swapping chaperone andnot as a hypothetical subunit-specific chaperone.

We here discovered self-subunit swapping of L-NHase, which isa thus-far unknown posttranslational maturation mechanism forcobalt incorporation. Clarification of the L-NHase biosynthesisincluding the investigation of cobalt incorporation into NhlAE andthe cysteine modification mechanism await further study.

Materials and MethodsDetailed descriptions of the purification of proteins, enzyme assay, determi-nation of molecular masses of proteins, denaturation of holo-�e2 and apo-�e2,renaturation of NhlE and the �-subunit, MALDI-TOF MS sequencing, CDmeasurement, UV-Vis measurement, NH2-terminal amino acid sequencing,and determination of cobalt ions in enzymes are given in Materials andMethods in SI Text.

Strains, Plasmids, Gene Cluster, and Culture Conditions. R. fascians DSM43985was used as the host for vector plasmid pREIT19, which was used for nhlBA,nhlBAE, nhlAE, nhl�AE, nhlE, and nhlBA-(�-A3G), nhlBA-(�-A3G)(�-R52A/R157A) and nhlBAE-(�-R52A/R157A) expression. Plasmid pLJK60 (10) carryingnhlBAE of R. rhodochrous J1 in a 3.1-kb KpnI fragment on the pK4 plasmid wasused for subcloning of the nhlBA and nhlBAE genes with primers B-up plusA-down, and B-up plus E-down (Table S1), respectively. See Materials andMethods in SI Text for details of the construction of pREIT-nhlBA, pREIT-nhlBAE, pREIT-nhlAE, pREIT-nhl�AE, pREIT-nhlE, pREIT-nhlBA-(�-A3G), pREIT-nhlBA-(�-A3G)(�-R52A/R157A), and pREIT-nhlBAE-(�-R52A/R157A), and theculture conditions.

ACKNOWLEDGMENTS. We thank Drs. K. Oinuma, H. Maseda, and R. Hirota forhelpful discussions; Dr. T. Abe and T. Sakashita for the amino acid sequenceanalysis; I. Sagawa, K. Yamamoto, and Y. Fukuta for the MALDI-TOF MS analysis;and M. Sakai for the ultracentrifugation. This work was supported in part by the21stCenturyCentersofExcellenceProgram(www.tara.tsukuba.ac.jp/�coe21/)ofthe Ministry of Education, Culture, Sports, Science, and Technology (MEXT), andby a Grant-in-Aid for Scientific Research from MEXT.

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