Discovery of Evolutionary Divergence of Biological Nitrogen Fixation and Photosynthesis: Fine Tuning of Biogenesis of the NifH and the ChlL by a Peptidyl-Prolyl Cis/Trans Isomerase

Problem statement: Despite the structural and functional similarities between the nitrogenase that performs biological nitrogen fixat ion reaction and the Dark Protochlorphyllide Oxidoreductase (DPOR) that performs chlorophyll-bio synthesis, attempts to substitute nitrogenasecomponents with DPOR-components have hitherto faile d. This investigation was undertaken to test if Chlamydomonas reinhardtii protochlorophyllide (Pchlide) reductase (ChlL) tha t shares some structural similarity with Nitrogenase Reductase (NifH) could complement the functions of NifH in biological nitrogen fixation of Azotobacter vinelandii. Approach: Genetic complementation studies were performed to test if the chlL gene and its mutants cloned under transcriptional control of ni H promoter (nifHp) in a broad-host range low copy plasmid pBG1380 co uld render a Nif + phenotype to NifHdeficient A. vinelandii strains. Results: Expression of ChlL could render Nif + phenotype to NifHdeficient A. vinelandii only in the absence of NifM, a nif-specific PPIase essential for biogenesis of NifH. The ChlL mutants Cys95Thr and Cys129Thr were unable to substitute for NifH. Thus, the conserved cysteine ligands of [4Fe-4S] cluster in C hlL are essential for successful substitution of NifH by ChlL. Since C-termini of NifH and ChlL demo nstrated the least similarity and Pro258, a substrate for the PPIase activity of NifM, is locat ed in the C-terminus of NifH, we posited that replacing the C-terminus of NifH with that of ChlL would render NifM-independence to NifH. The NifH-ChlL chimera could support the growth of NifHand NifM-deficient A. vinelandii in nitrogen limiting conditions implying that it has acquired N ifM-independence. Conclusion/Recommendations: Collectively, these observations suggest that NifM , an evolutionarily conserved nif-specific PPIase, could have contributed to the functional divergence of biological nitrogen fixation and photosynthesis dur ing evolution by virtue of its ability to exert opposing effects on structurally similar substrates , ChlL and NifH.


INTRODUCTION
Functional divergence of biological nitrogen fixation and photosynthesis, the two fundamental biological processes that sustain life on earth, is still an enigma. Structural and functional similarities exist between nitrogenase that performs the biological nitrogen fixation reaction and Dark Protochlorphyllide Oxidoreductase (DPOR) that performs reduction of protochlorophyllide to chlorophyllide during chlorophyll-biosynthesis (Brocker et al., 2008;Gavini et al., 2006;Georgiadis et al., 1992;Sarma et al., 2008;Tezcan et al., 2005;Watzlich et al., 2009;Yamamoto et al., 2009;Yamazaki et al., 2006a;Nomata et al., 2006a;. Both nitrogenase and DPOR are oxygen sensitive twocomponent systems. However, attempts to substitute nitrogenase components NifH or NifDK by DPOR components have not been successful thus far. vinelandii NifH and C. reinhardtii ChlL using. ClustalW (http://www.clustal.org/). The conserved cysteine ligands are shown in light yellow box. The ATP binding site is shown in gray box. The four proline residues that are conserved between the NifH, BchL and the ChlL are marked by open box. Three more additional proline residues that are conserved among NifH peptides are highlighted in light gray and the additional proline residues that are conserved among BchL and ChlL (shown in Fig. 2) are highlighted in dark gray Nitrogenase is dependent on a multitude of nif-specific accessory proteins for its maturation and assembly (Brocker et al., 2008;Gavini et al., 2006;Georgiadis et al., 1992;Yamazaki et al., 2006b;Betancourt et al., 2008;Chen et al., 1994;Christiansen et al., 2001;Curatti et al., 2007;Gavini and Burgess, 1992;Gavini et al., 1994;Finan, 2002;Howard et al., 1989;1986;Howard and Rees, 1996;Jacobson et al., 1989a;1989b;Lei et al., 1999;1998;Peters and Szilagyi, 2006;Petrova et al., 2002;Rubio and Ludden, 2005;Robinson et al., 1987). The DPOR component analogous to the nitrogenase component NifH is the BchL/ChlL protein encoded by the bchL/chlL genes of the photosynthetic bacteria.
Biogenesis of functional NifH is dependent on nif-accessory protein NifM in its natural system or in heterologous system (Finan, 2002;Howard et al., 1986;Jacobson et al., 1989a;Petrova et al., 2002). The NifM is a peptidyl-prolyl cis/trans isomerase (PPIase) belonging to the Parvulin family (Edlich and Fischer, 2006;Rahfeld et al., 1994). We have proposed that the NifM-mediated cis-to trans isomerization of one or more of the seven conserved prolines is needed for the generation of the functional NifH (Finan, 2002 (Fig. 1) that are part of the nine conserved prolines among the ChlL peptides (Fig. 2). Additionally, superimposing a predicted model of ChlL onto the NifH template (PDB ID: 1NIP) (Georgiadis et al.,1992), using Swiss PDB (Deep View) protein modeling software (Fig. 3) shows that the ChlL protein model thus generated has marked resemblance to the NifH protein. crystallographic structure of the NifH peptide from A. vinelandii (Georgiadis et al., 1992)  Since the ChlL protein showed such high structural similarity to the NifH protein, we hypothesized that ChlL would substitute for NifH in A. vinelandii and that NifM may play a role in modulating the functional properties of the NifH-like proteins such as ChlL/Bchl. The purpose of this study was to determine whether ChlL could substitute for NifH in biological nitrogen fixation in presence and absence of functional NifM.

MATERIALS AND METHODS
Construction of plasmid pBG2400: An 879bp DNA fragment encoding the ORF of chlL flanked by EcoRV and HindIII restriction enzyme sites was generated by PCR amplification using C. Reinhardtii chromosomal DNA as template and cloned into the pCR2.1 TOPO vector to create plasmid pBG1382 (Suh, 2002). This fragment was then subcloned into the EcoRV and HindIII digested broad-host range expression vector pBG1380 that contained the nifHp (Gavini et al., 2006) to generate pBG2400. Thus, the plasmid pBG2400 had the chlL gene under the transcriptional regulation of the nifHp.

Construction of NifH-ChlL chimera:
The 873bp DNA fragment encoding nifH ORF was PCR amplified using pDB6 (Jacobson et al., 1989b) as the template and initially cloned into the pCR2.1 TOPO vector. The EcoRV-HindIII fragment encoding nifH ORF was subcolned into EcoRV-HindIII digested pBG1380 to generate pBG2434 that carries nifH gene under the transcriptional control of nifH promoter (nifHp). The129bp region that carries the last 42 amino acids at the C-terminus of NifH was removed via Sal1 digestion. Next, the DNA containing the last 55 codons of the chlL was PCR-amplified using a 5' primer that carries a SalI site (5'-GTCGACAATTCTACAGTAGGAGTGTC-3') and a 3' primer with a HindIII site (5-AAGCTTTTAAATTTTAAGATAGAAATC-3'). The resultant PCR product encoding the C-terminal region of ChlL protein (55 amino acids at the C-terminal end) was cloned into the SalI-HindIII digested pBG2434 (carrying the N-terminus of nifH) to generate a nifH-chlL chimeric gene in which the DNA encoding the C-terminal region of NifH (bp745-873) was replaced by the DNA encoding C-terminal region of ChlL (bp718-882).

RESULTS AND DISCUSSION
ChlL can substitutes for NifH in biological nitrogen fixation reaction only in the absence of NifM: Two NifH-deficient A. vinelandii strains, one NifMpositive (nifM + A. vinelandii DJ54 Robinson et al., 1987) and one NifM-negative (nifM::kan A. vinelandii BG98 (Gavini et al., 2006) respectively, were used to test the ability of the ChlL to substitute for the NifH in nitrogen fixation reaction by A. vinelandii. Both strains were transformed with pBG2400 that carries the C. reinhardtii chlL gene cloned under the transcriptional regulation of the nifHp and the ability of the transformants to grow under nitrogen limiting conditions was assessed as follows.  (Strandberg and Wilson, 1968) that imits expression of alternate nitrogenases (Betancourt et al., 2008).
It was found that pBG2400 could not render a Nif + phenotype to the A. vinelandii DJ54 (Fig. 4a), but it rendered Nif + phenotype to the A. vinelandii BG98 (Fig. 4b).
Since the difference between the two strains is that DJ54 has an intact nifM, whereas BG98 has a disrupted nifM, we concluded that the ChlL could restore nitrogenase activity in the absence of functional NifHbut only if NifM was also absent. In summary, the ChlL can replace the NifH-function in biological nitrogen fixation by A. vinelandii. However, the NifM, the accessory PPIase essential for biogenesis of functional NifH, has a negative effect on the compensatory ability of ChlL. This interpretation is consistent with the observations that (a) the chlL gene could not complement the ∆nifH of nifM + A. vinelandii DJ54 and (b) the BchL protein (similar in structure and function to the ChlL) isolated from nifM + A. vinelandii was unable to substitute for the NifH protein in nitrogenase assay (Sarma et al., 2008).

Cys95 and Cys129 of the ChlL are required for substitution of NifH by ChlL in biological nitrogen fixation reaction:
The cysteine ligands Cys97 and Cys132 of the NifH peptide are conserved in the ChlL peptide (Cys95 and Cys129 of the ChlL peptide respectively; Fig. 1). Replacing these conserved cysteines of NifH impairs its function (Howard et al., 1989). We posited that the Cys95 and Cys129 of the ChlL have a similar role in the ability of the ChlL to participate in nitrogen fixation reaction. Therefore we generated ChlL mutants Cys95Thr and Cys129Thr. In both mutants a TGT to ACC conversion was made that resulted in the codon change TGT (Cys) to ACC (Thr) and created a new PinA1 restriction enzyme site that facilitated identification of these mutants. Locations of the mutations were confirmed by nucleotide sequencing. As shown in Table 1, neither of the mutant chlL genes was able to support the growth of nifM -NifH-deficient A. vinelandii BG98 on BNmedium. Growth of the NifH-deficient nifM -A. vinelandii strain BG98 carrying the parental plasmid pBG1380 (marked 2) and pBG1380-derivative expressing the nifH-chlL chimeric gene (marked 3) on BNmedium is shown. Wild type A. vinelandii (marked 1) served as control. Thus, the NifH-ChlL chimera could support growth of nifM -A. vinelandii strain BG98 in nitrogen limiting conditions indicating that replacement of the Cterminal region of the NifH with that of the ChlL resulted in partial relief from NifM-dependence. Experiments were repeated at least six times These observations suggested that the Cys95 and Cys129 of the ChlL could play roles analogous to that of Cys97 and Cys132 of NifH in stabilizing the [4Fe-4S] cluster of ChlL.
A nifH-chlL chimera could render Nif + phenotype to A. vinelandii BG98: As shown in Fig. 1 and 3, the Ctermini of the NifH and the ChlL are highly dissimilar. Therefore, the fact that NifH is not functional in the absence of NifM while ChlL is not functional in the presence of NifM might be traced to this region. We have shown previously that the Pro258 located in the C-terminus of the NifH is one of the substrates for the PPIase activity of NifM (Gavini et al., 2006).
Because the C-terminus of the ChlL is dissimilar to that of the NifH and the ChlL could substitute the NifH in the absence of the NifM in nitrogen fixation, it is conceivable that the C-terminus of the ChlL would render NifM-independence to the NifH. To test this idea, we analyzed the effect of replacing the C-terminus of NifH with that of ChlL. The DNA encoding the Cterminal region of NifH (bp745-873) was replaced by the DNA encoding C-terminal region of ChlL (bp718-882) to construct the nifH-chlL chimeric gene. Therefore, the resulting NifH-ChlL chimera did not contain Pro258 of the NifH. Amino acid sequence of the NifH-ChlL chimera is shown in Fig. 5a. A. vinelandii BG98 transformants expressing the nifH-chlL chimeric gene were capable of growing on BNmedium (Fig. 5b). Thus, the nifH-chlL chimera could render partial NifMindependence to A. vinelandii BG98.

CONCLUSION
Our results show that the NifM, a NifH-specific PPIase that is essential for biogenesis of the NifH protein, has a role in disabling structurally similar ChlL from participating in the biological nitrogen fixation reaction. Significance of PPIase-substrate interactions are particularly highlighted in many pathological conditions. For example, overexpression of human Pin1 is implicated in the formation of Lewy bodies in Parkinson's Disease, while the same protein has a beneficial effect in Alzheimer's disease, since it regulates amyloid precursor protein processing and amyloid beta production (Pastorino et al., 2006;Ryo et al., 2006). Similarly, Macrophage Infectivity Potentiators (MIPs) are PPIases expressed by bacterial pathogens, however, they interact with host-cell proteins and alter their functions to establish infection (Kohler et al., 2003). These examples show that the molecular interactions between PPIases and proteins that share structural similarity to their natural substrates result in pathogenesis. The example that has emerged from this study is that of a PPIase which could have contributed to the functional divergence of two fundamental biological processes (nitrogen fixation and photosynthesis) during evolution. This is because this PPIase prototype (NifM) would render functionality to one substrate (NifH) and hinder the function of the other structurally similar substrate (ChlL) so that nitrogen fixation is favored under conditions that lead to NifM expression (such as nitrogen limitation). These findings represent a unique example of an accessory protein playing a vital part in the evolutionary divergence of biological processes.
The observation that ChlL mutants Cys 95Thr and Cys129Thr were unable to substitute for NifH further extends the structure-function similarity of the NifH and ChlL related to their mechanistic involvement in nitrogen fixation. Although the structure of ChlL is not yet solved, these observations strengthen the similarities in the role of Cys ligands of the (4Fe-4S) cluster of ChlL in electron transfer by the ChlL to that of the NifH. On the other hand, our studies also highlight the dissimilarity of the C-termini of the NifH and the ChlL. The C-terminus of the NifH is involved in the NifM-dependence of the NifH due to the presence of Pro258 (Gavini et al., 2002). In contrast, the C-terminus of the ChlL could render NifMindependence to the NifH as shown by the functional NIfH-ChlL chimera (Fig. 5). It is conceivable that a protein similar to the NifH-ChlL chimera could have served as a common ancestor for the NifH and the ChlL before the functional divergence of biological nitrogen fixation and photosynthesis during evolution.