The Lotus japonicus Genome

5.4.2 Genes Involved in Nitrogen
Fixation

There are 28 conserved genes probably involved
in nitrogenfixation and related reactions. These
include two copies of the regulatory genenifA,
genes encoding the nitrogenase enzyme and
genes specifically involved in the synthesis of the
complex metallo-clusters, electron transport, and
energy conversion processes (Kaneko et al.
2000 ; Sullivan et al. 2002 ; Kasai-Maita et al.
2013 ). The regulatory hierarchy of the two NifA
proteins is described below.
The first three genes of the nifHDKENX
cluster encode structural proteins of nitrogenase
enzyme complex that consists of two compo-
nents. The terminal enzyme, dinitrogenase or
MoFe protein, responsible for reduction of
molecular nitrogen (N 2 ) is a hetero-tetramer
(α 2 β 2 ) of NifD and NifK proteins that contains
the metallo-clusters, iron–molybdenum cofactor
(FeMo-co) and P cluster (Dixon and Kahn 2004 ;
Rubio and Ludden 2008 ). Reducing equivalents
and free energy to carry out the reaction are
transferred to dinitrogenase from dinitrogenase
reductase or Fe protein that is a homodimer of
NifH protein containing a 4Fe–4S cluster and
binding sites for Mg–ATP. The transfer of an
electron is achieved by transient association
between reduced ATP-bound Fe protein and
MoFe protein. The hydrolysis of ATP by Fe
protein causes a conformational change and is
coupled to electron transfer from the 4Fe–4S
cluster to P cluster in MoFe protein and

dissociation. Electrons are then transferred from

P cluster to FeMo-co where the catalytic reaction

takes place.

FeMo-co is a unique cofactor only found in

molybdenum nitrogenase. This cofactor consists

of an inorganic moiety of Mo–Fe 7 – S 9 and an

organic moiety of R-homocitrate (Rubio and

Ludden 2008 ). Products of nifEN are central

scaffold of FeMo-co synthesis and transfer to

nifDKproduct. NifH protein is also believed to

function in FeMo-co synthesis with NifE and

NifN protein complex. Most othernifgenes have

functions to supply or process constituents of

FeMo-co. ThenifSproduct is a cysteine desul-

furase to supply sulfur to assemble transient

Fe–S clusters on a scaffold protein generally

encoded bynifU.NonifUhomologue is located

on the threeM. lotisymbiosis islands; however,

there is one homologue,mll0920, on the core

chromosome of strain MAFF303099, whose

product might function as NifU scaffold. The

Fe–S cluster on NifU is then transferred to

another scaffold, a dimeric product ofnifB,that

utilizesS-adenosyl methionine to assemble the

FeMo-co precursor. The precursor on NifB is

then transferred to a NifEN scaffold with the help

of the product ofnifX. On the NifEN scaffold,

molybdenum is donated by the product ofnifQ

(Rubio and Ludden 2008 ), whileR-homocitrate

is donated by the product ofnifV(Hoover et al.

1987 ). AlthoughnifQis present on theM. loti

symbiosis islands, no nifV that encodes the

homocitrate synthase is found, not only on the

three symbiosis islands but also on the core

Table 5.1 (continued)

3-methylaspartate

ammonia-lyase

maaL mlr6095 ML0187 mln268

2-amino-3-ketobutyrate

CoA ligase

kbl mll9204 (on

pMLa)

ML0254 N. D.

Cysteine synthase cycM N. D. mll9227on

(pMLa)

ML0212 N. D.

Phosphate metabolism

Phosphonate degradation phnG–phnH–

phnI–phnJ–

phnK–phnM–

phnL

N. D. mlr9277–

mlr9286 (on

pMLa)

ML0321–

ML0315

N. D.

N. D.: Not Detected

50 K. Saeki and C.W. Ronson