Possible Function for NifM

As mentioned above, the K. pneumoniae nifM gene product shares very low homology with the products of the nifM genes from Azotobacter. However, some significant homology is present in the carboxyl terminal region of these proteins suggesting that the observed functional similarity (interspecies complementation) is probably confined to this region. We compared the carboxyl terminal region of the nifM genes and generated a consensus sequence which was then compared to the conceptually translated nucleotide sequence databases. This comparison showed that the carboxyl terminal region of the NifM proteins shares significant homology with the family of proteins called peptidyl-prolyl cis/trans isomerases (Figure 2).

Peptidyl-prolyl cis/trans isomerases (PPIases) are thought to be involved in assisting protein folding (Fisher, 1994). These proteins catalyze the cis/trans isomerization of the peptidyl-prolyl peptide bond in oligopeptides and proteins - a rate-limiting step in the process of protein folding essential for generating functional proteins. Some denatured proteins regain their native conformation within milliseconds to seconds; whereas, others refold very slowly and the time range varies from minutes to hours. The slow conformational changes arise from the well-known derealization of electrons in the amide bond and are even more pronounced if additional steric constraints are imposed by the proline ring. In contrast to C(0)NH bonds, the prolyl-peptide bonds exist in the two lowest energy conformational forms - cis (w = 0°) and trans (w = 180°) - which are thermodynamically comparable. Therefore, polypeptides with n proline residues can exist in 2" isomeric forms (Figure 3). When the proteins form three-dimensional structures, the cis- and trans-

isomers in these structures are no longer of equal energy and the less preferred isomer is not likely to exist in the folded chain. According to the proline hypothesis, it is assumed that the slow folding forms of the unfolded protein possess non-native isomers of the peptide bonds between proline and another residue and that the crucial steps in the refolding of the slow folding molecules are limited in rate by the slow isomerization of such incorrect proline-peptide bonds. PPIases have been demonstrated to catalyze the cisltrans isomerization of these peptidyl-prolyl bonds and, thus, to enhance the rate of refolding of the slow folding forms of denatured proteins.

Domain 1

Domain 2

NifM Consensus

--h-l-t---d--el-r--------fa--a-

-hs-CD--a---q-lq----

A. vinelandii

NifM

KAHILVTINEDFPELKRLRGK PERFAEQAMKHSECPT-AMQGGLLGEWP

A.chroococcum NifM

ARHILVTINEDFPELKRLRGK PERFAEQAMKHSECPT-AMQGGLLGEWP

K. pneumoniae

NifM

TRHLLLTVDNDR-ELYRQINASRDAFAPLAQRHSHCPS -ALEEGRLGWISR

B.coli PpiC

ALHILVKEEKLALDLLEQIKNGAD-FGKLAKKHSICPS-GKRGGDLGEFRQ

B. subtillis

PrsA

ASHILVADKKTAEEVEKKLKKGEK-FEDLAKEYSTDSS - ASKGGDLGWFAK

B.coli SurAn

LSHILIPLPENPTSIVDQARNGAD-FGKLAIAHSADQQ-ALNGGQMGWGRI

E.coli SurAc

ARHILLKPSPIMTDAADIESGKTT-FAAATKEFSQDPVSANQGGDLGWATP

B.subtillis

Orf

IRHIWKDEEEAREVLKELKGGSS -FEAVAAERSTDRYTSPYGGDLGFVTE

L. la PrtM

VQHILTSDEDTAKQVISDLAAGKD-FAMLAKTDSIDTATKDNGGKISFELN

EST human

FQSPASQFSDC-S SAKARGDLGAFSK

D.disc. ORF

GDP - - RQRGGDLGWAPA

A.thaliana PPIASE

FEEVATRVSDC-S SAKRGGDLGSFGR

Domain3

Consensus

--Q-lvP-1-

--lf--a---Is-----s--cr-h-l-ce---

NifM-A. v.

--GTLYPELDACLFQMARGELSP-VLESPIGFHVLYCESVS

[P14890]

NifM-A.c.

--GTLYPELDACLFQMARGELSP-VLESPIGFHVLFCESVS

[P23119]

NifM-K.p.

- -GLLYPQLETALFSLAENALSL - PIASELGWHLLWCEAIR

[P08534]

PpiC-E. c.

- -GQMVPAFDKWFSCPVLEPTG- PLHTQFGYHIIKVLYRN

[M87049]

PrsA-B.s.

-EGQMDETFSKAAFKLKTGEVSD-PVKTQYGYHIIKKTEER

[P24327]

SurAn-E.c.

--QELPGIFAQALSTAKKGDIVG-PIRSGVGFHILKVNDLR

[S40574p]

SurAc-E.c.

--DIFDPAFRDALTRLNKGQMSA-PVHS S FGWHLIELLDTR

[S40574p]

Orf-B.s.

ASDNIPSAYIEEAKTLKEDEWSQEPIKVSNGYAIIQLKEKL

[D26185_135g]

PrtM-L.1.

- NKTLDATFKDAAYKLKNGD YTQTPVKVTDGYEVI KM INHP

[Q02473]

EST human

--GQMQKPFEDPWFARRTGEMSG-TVFTDSGIHVIVRTE

[M86110g]

Orf-D.d.

- TNYVQPFAEAVTKLKKGQLVDK- PVQTQFGWHVIQVDDTR

[X70280]

PPIASE-A.t.

GQMQKPFEEATYALKVGDISD IVDTDSGVHII

[F13919]

Figure 2. Comparison of the consensus sequence of the NifM protein to the amino acid sequences of the peptidyl-prolyl cisltrans isomerases. The three domains that share high homology are identified by overlining and are numbered arbitrarily. The fully conserved residues in each peptide are shown in bold letters.

/—

—V

R. / -

>

o J

Figure 3. Differences in the structure between the cis and trans isomers of a peptide bond N-terminal to proline.

Figure 3. Differences in the structure between the cis and trans isomers of a peptide bond N-terminal to proline.

PPIases are expressed ubiquitously and the genes encoding these proteins have been identified in plants and animals as well as in lower eukaryotes and bacteria (Fisher 1994 and references therein). These proteins are also called immunophilins since they bind immunosuppressive drugs such as cyclosporin, FK506 and rapamycin. The two major families of PPIases are called the cyclophilins (cyclosporin-binding proteins) and FKBPs (FK506-binding proteins that also bind rapamycin). The two families have little sequence similarity to each other. However, within each family there are regions containing amino acids that have been highly conserved throughout evolution. These highly conserved regions span about 100 residues for cyclophilins and about 80 residues for FKBPs. In E. coli, two genes encoding cyclophilins and two genes encoding FKBP-related proteins have been identified so far. Apart from these genes, a new gene was recently isolated from E. coli that encodes a protein with PPIase activity that is not inhibited by either cyclosporin or FK506. This protein, which does not show any significant homology to the PPIases belonging to either the cyclophilin or the FKBP family, has a very low molecular mass and was called PpiC or Parvulin (Rudd et al. 1995). However, comparison of this protein with the predicted amino acid sequences in the database showed three regions of significant amino acid similarity between the PpiC protein and the Bacillus subtilis lipoprotein PrsA, the E. coli protein SurA and the NifM proteins (Figure 2). Based on this comparison, it has been suggested that the PpiC protein and the proteins that share amino acid similarity with PpiC define a new family of PPIases.

Thus, our comparison studies as well as others (Rudd et al. 1995) indicate that one of the possible functions of the NifM protein is to assist in the proper folding of the Fe-protein by catalyzing the conformational interconversions by the cisltrans isomerization of the peptide bond N-terminal to the proline residues present in this peptide. An examination of the consensus peptide sequence of the Fe-protein derived by comparing different Fe-proteins indeed shows the existence of seven fully conserved proline residues (Figure 1). Therefore, it is reasonable to assume that the stability and activity of the protein will largely depend upon the appropriate conformation of the peptidyl-prolyl bonds present in this protein. To test this idea, we proceeded to overexpress and purify the wild type K. pneumoniae and A. vinelandii NifM proteins and analyze these proteins for PPIase activity. Our analysis made use of the standard protease-coupled assay that is used to determine PPIase activity (Fisher 1994) and allowed us to assess the ability of the NifM protein to catalyze the cisltrans isomerization of the peptidyl-prolyl bond in an oligopeptide. The results suggested that the extent of PPIase activity that the NifM exhibited was comparable to that of PpiC of E. coli, a PPIase that shares homology with NifM.

In order to understand the NifM protein's role in modifying the stability and activity of the Fe-protein, we have also investigated existence of direct interaction between the NifM protein and the Fe-protein using the 'MATCHMAKER Two-Hybrid' yeast-based genetic assay as shown in Figure 4. Our preliminary results indicated existence of direct interaction between the Fe-protein

(translationally fused to GAL4 DNA binding domain [GAL4BD]) and the NifM protein (translationally fused to GAL4 activation domain [GAL4AD]) in yeast two-hybrid protein-protein interaction assay.

Cotransform ' Yeast strain Y190

Cotransform ' Yeast strain Y190

Cells can synthesize fusion proteins

Activation Domain

Cells can synthesize fusion proteins

Make selection on SD trp " leu " medium

NifM-NifH interaction brings G:il4 domains into close pro\iniit>

NifM-NifH interaction brings G:il4 domains into close pro\iniit>

BINDING DOMAIN

Figure 4. The general strategy used for analyzing the interaction between the Fe-protein and the NifM protein by using the 'MATCHMAKER Two-Hybrid' yeast-based genetic assay.

BINDING DOMAIN

Figure 4. The general strategy used for analyzing the interaction between the Fe-protein and the NifM protein by using the 'MATCHMAKER Two-Hybrid' yeast-based genetic assay.

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