Fusobacterium nucleatum is a common oral bacterium
and a major producer of H2S, a toxic gas linked to the
pathogenesis of periodontal disease. The bacterium encodes a fold
type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220
or lanthionine synthase (LS), that generates H2S and l-lanthionine (a component of the peptidoglycan layer) through
β-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis
that LS elicits catalytic promiscuity as demonstrated for other fold
type II PLP-dependent homologues, namely, O-acetylserine
sulfhydrylase (OASS) and cystathionine β-synthase (CBS). Like
OASS, LS can assimilate H2S by catalyzing the β-replacement
of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is
slower than that of other studied OASS enzymes due to slower conversion
to the α-aminoacrylate intermediate. Similar to yeast and human
CBS, LS can generate H2S and l-cystathionine through
β-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference
for forming H2S and l-lanthionine through the
condensation of 2 equiv of l-cysteine. Sequence alignment
of LS with other CBS and OASS enzymes and inspection of the LS crystal
structure in the external aldimine state with l-lanthionine
reveal that LS possesses a unique loop that engages in hydrogen-bond
contact with the product, providing a structural rationale for the
enzyme’s catalytic preference for H2S and l-lanthionine biosynthesis.
A number
of species within the Fusobacteriaceae family of
Gram-negative bacteria uniquely encode for an ornithine
decarboxylase/arginase (ODA) that ostensibly channels l-ornithine
generated by hydrolysis of l-arginine to putrescine formation.
However, two aspartate residues required for coordination to a catalytically
obligatory manganese cluster of arginases are substituted for a serine
and an asparagine. Curiously, these natural substitutions occur only
in a clade of Fusobacterium species that inhabit the oral cavity.
Herein, we expressed and isolated full-length ODA from the opportunistic
oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components.
The crystal structure of the arginase domain reveals that it adopts
the classical α/β arginase-fold, but metal ions are absent
in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated
arginase domain. However, engineering of the complete metal coordination
environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency
for l-arginine was low (60–100 M–1 s–1). Full-length ODA and the isolated ODC component
were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but k
cat/K
M of l-ornithine was ∼20-fold higher compared to l-arginine.
We discuss environmental conditions that may have led to the natural
selection of an inactive arginase in the oral associated species of Fusobacterium.
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