proteins with high sequence homology to NifBMa and NifBMt (over
300 proteins with a homology of higher than 70% over a range of
85% of the sequence). The organisms expressing these truncated
NifB homologs (60% methanogenic organisms and 40% non-
methanogenic organisms) are widespread across the microbial
biorealm; many of them are not nitrogen-fixing organisms, sug-
gesting that the NifB proteins in these organisms carry out other
functions that are yet to be identified. Sequence alignment of 45
closest matches of NifB homologs revealed the presence of the
canonical radical SAM domain, as well as the same, conserved Cys
and His residues as those identified in NifBMa and NifBMt, which
potentially serve as FeS cluster-binding domains (Fig. S6). A new
proposed based on this finding, which potentially specializes in
radical SAM-based assembly of complex metallocenters.
Other than enabling the classification of a distinct subset
of RSMTs, the successful identification of NifBMa or NifBMt
as functional homologs of NifBAv is exciting, as it opens up
new avenues to study the structure and mechanism of the NifB
protein, both of which remain relatively uncharacterized and
promise to reveal completely unprecedented chemical reactions
catalyzed by biological systems. The fact that both methanogen
NifB homologs can be expressed alone in E. coli as soluble, intact
proteins permits structural and biochemical analysis of NifB
without interference of its protein partner and associated metal
centers, a feat that has not been achieved so far through inves-
tigations of the NifEN-B fusion protein; moreover, it suggests
the possibility to express a truncated version of NifBAv in E. coli,
which can then be used for comparative studies with its newly
identified homologs in methanogens to shed light on the struc-
ture–function relationship of this important protein family.
Novel mechanistic insights could be gained by studying these
proteins side by side, and species-dependent differences revealed
by these studies—such as a shift toward a higher SAH/5′-dAH
ratio in the cases of NifBMa and NifBMt—could be explored to
reveal differential mechanisms used by the NifB homologs to
abstract hydrogen from the substrate-bound methyl group or
identify additional functions of these proteins as methyltransferases
in their native hosts. Additionally, important “snapshots” of carbide
insertion pathway could be captured by mixing and matching the
NifB protein from one organism with assembly components from
another, which may back up certain intermediates on NifB due to a
less efficient transfer of L clusters from NifB to its downstream
assembly partner. All in all, the NifB homologs reported in this
work provide a brand new tool in addition to the NifEN-B fusion
protein (23) for further characterization of the distinctive methyl
transfer and conversion to an iron-bound carbide by NifB, which is
crucial for the unveiling of a chemically unique and biologically
important reaction pathway.
Materials and Methods
Unless noted otherwise, all chemicals and reagents were obtained from Fisher
Scientific or Sigma-Aldrich. Cell growth, protein purification, iron/sulfur re-
constitution, molecular mass determination, iron determination, SAM cleavage
assays, EPR analysis, cluster maturation assays, carbon-14 tracing experiments,
cluster extraction, and PTM analysis were performed as described. See SI Materials
Genes encoding the NifB homologs from M. acetivorans (NifBMa) and
M. thermoautotrophicum (NifBMt) were codon optimized for E. coli expres-
sion and synthesized and cloned into the BamHI site of pET-3b and the NdeI
site of pET-14b, respectively (GenScript USA). A short sequence encoding a
6xHis tag was inserted at the 5′-end of the gene encoding NifBMa before the
cloning of this sequence into pET-3b, whereas the gene encoding NifBMt
was placed behind a vector-derived sequence encoding a 6xHis tag when
it was cloned into pET-14b. Each of these constructs was then cotransformed
with a plasmid harboring iscSUA and hscABfdx genes—an ensemble of
genes encoding Fe/S cluster assembly proteins (24–28)—into the E. coli strain
BL21(DE3), resulting in strains overexpressing His-tagged forms of NifBMa
(strain YM114EE) and NifBMt (strain YM127EE) upon induction by isopropyl
β-D-1-thiogalactopyranoside (IPTG). The plasmid carrying iscSUA and hscABfdx
genes was a generous gift from S. Leimkhüler (University of Potsdam, Pots-
dam, Germany). The gene encoding NifX from A. vinelandii (NifXAv) was PCR
amplified from the genomic DNA using a pair of primers (forward primer:
5′-ATGGTAGGTCTCAAATGTCCAGCCC GACCCGACAATTG-3′; reverse primer:
5′-ATGGTAGGTCTCAGCGCTTTCGTCCCAGCCTTCGGCGG-3′) and subsequently
cloned into the BsaI site of pASK-IBA3 (IBA). This construct was transformed
into the E. coli strain BL21-CodonPlus, resulting in a strain expressing a C
terminus strep-tagged form of NifXAv (strain YM300EE).
ACKNOWLEDGMENTS. We thank Prof. Markus Ribbe [University of Califor-
nia, Irvine (UCI)] for helpful discussion and technical support of biochemical
experiments related to the NifEN-B′ fusion protein. This work was supported
by UCI startup funds and a Hellman Fellowship (to Y.H.).
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