SbnI is a free serine kinase that generates O-phospho-L-serine for staphyloferrin B biosynthesis in Staphylococcus aureus

Article abstract of DOI:10.1074/jbc.RA118.001875

Staphyloferrin B (SB) is an iron-chelating siderophore produced by Staphylococcus aureus in invasive infections. Proteins for SB biosynthesis and export are encoded by the sbnABCDEFGHI gene cluster, in which SbnI, a member of the ParB/Srx superfamily, acts as a heme-dependent transcriptional regulator of the sbn locus. However, no structural or functional information about SbnI is available. Here, a crystal structure of SbnI revealed striking structural similarity to an ADP-dependent free serine kinase, SerK, from the archaea Thermococcus kodakarensis. We found that features of the active sites are conserved, and biochemical assays and³¹P NMR and HPLC analyses indicated that SbnI is also a free serine kinase but uses ATP rather than ADP as phosphate donor to generate the SB precursor O-phospho- L-serine (OPS). SbnI consists of two domains, and elevated B-factors in domain II were consistent with the open-close reaction mechanism previously reported for SerK. Mutagenesis of Glu²⁰ and Asp⁵⁸ in SbnI disclosed that they are required for kinase activity. The only known OPS source in bacteria is through the phosphoserine aminotransferase activity of SerC within the serine biosynthesis pathway, and we demonstrate that an S. aureus serC mutant is a serine auxotroph, consistent with a function in L-serine biosynthesis. However, the serC mutant strain could produce SB when provided L-serine, suggesting that SbnI produces OPS for SB biosynthesis in vivo. These findings indicate that besides transcriptionally regulating the sbn locus, SbnI also has an enzymatic role in the SB biosynthetic pathway.

Full text of DOI:10.1074/jbc.RA118.001875

JBC Papers in Press. Published on February 26, 2018 as Manuscript RA118.001875
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.001875
SbnI is a free serine kinase that generates O-phospho-L-serine for staphyloferrin B biosynthesis in
Staphylococcus aureus
1
2
1
3
Meghan M. Verstraete , Cecilia Perez-Borrajero , Kirstin L. Brown , David E. Heinrichs , Michael
1
E. P. Murphy *
1
2
From the Department of Microbiology and Immunology, Genome Sciences and Technology Program
3
Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada; Department
of Microbiology and Immunology, University of Western Ontario, London, ON, N6A 5C1, Canada
Running title: SbnI serine kinase activity and role in SB biosynthesis
*
To whom correspondence should be addressed: Michael E. P. Murphy, Department of Microbiology and
Immunology, 2350 Health Sciences Mall, The University of British Columbia, Vancouver, BC, Canada,
V6T 1Z3; Telephone: (604) 822-8022; E-mail: michael.murphy@ubc.ca
Keywords: Staphylococcus aureus (S. aureus), siderophore, kinase, crystal structure, kinetics, O-
phospho-L-serine, staphyloferrin, virulence factor
ABSTRACT
Staphyloferrin B (SB) is an iron-chelating
serine biosynthesis. However, the serC mutant
strain could produce SB when provided L-serine,
suggesting that SbnI produces OPS for SB
biosynthesis in vivo. These findings indicate that
besides transcriptionally regulating the sbn locus,
SbnI also has an enzymatic role in the SB
biosynthetic pathway.
siderophore produced by Staphylococcus aureus
in invasive infections. Proteins for SB
biosynthesis and export are encoded by the
sbnABCDEFGHI gene cluster, in which SbnI, a
member of the ParB/Srx superfamily, acts as a
heme-dependent transcriptional regulator of the
sbn locus. However, no structural or functional
information about SbnI is available. Here, a
crystal structure of SbnI revealed striking
structural similarity to an ADP-dependent free
serine kinase, SerK, from the archaea
Thermococcus kodakarensis. We found that
features of the active sites are conserved, and
Staphylococcus aureus is a prominent
human pathogen that also asymptomatically
colonizes a proportion of the human population
(1, 2). Though colonization is typically not
harmful to the host, S. aureus is frequently
associated with minor skin and soft tissue
infections. In more serious cases, it is capable of
breaching host innate immune responses to gain
access to deep tissues causing more severe and
invasive infections including endocarditis,
osteomyelitis, and necrotizing pneumonia (2, 3).
Successful iron uptake from the human
host is integral to infection and pathogenesis of S.
aureus and most other microbial pathogens (4, 5).
Iron is reactive and tightly regulated in the human
body to restrict toxicity and to limit
bioavailability to invading microbial pathogens as
a type of innate, nutritional immunity (6). As
such, iron in the mammalian host is found either
within heme and bound to hemeproteins,
31
biochemical assays, and P NMR and HPLC
analyses indicated that SbnI is also a free serine
kinase but uses ATP rather than ADP as
phosphate donor to generate the SB precursor O-
phospho-L-serine (OPS). SbnI consists of two
domains, and elevated B-factors in domain II
were consistent with the open-close reaction
mechanism previously reported for SerK.
Mutagenesis of Glu20 and Asp58 in SbnI
disclosed that they are required for kinase activity.
The only known OPS source in bacteria is through
the phosphoserine aminotransferase activity of
SerC within the serine biosynthesis pathway and
we demonstrate that an S. aureus serC mutant is a
serine auxotroph, consistent with a function in L-
1

SbnI serine kinase activity and role in SB biosynthesis
intracellularly bound to proteins as free ions or
iron-sulfur clusters, stored in ferritin, or
extracellularly complexed with glycoproteins like
transferrin (7, 8). S. aureus has evolved several
mechanisms to counter iron restriction and exploit
a variety of host iron sources. Iron uptake
strategies employed include heme uptake via the
iron-responsive surface determinant (Isd) system
and through synthesis and secretion of two iron-
chelating siderophores, staphyloferrin A (SA) and
staphyloferrin B (SB) (9–11). Siderophores are
small molecules with high iron affinity capable of
outcompeting extracellular host iron-binding
proteins for ferric iron. Iron-bound siderophores
are selectively imported by dedicated surface
receptors and cognate ABC transporters, HtsABC
and SirABC for SA and SB, respectively (12).
SA and SB have both been implicated in
pathogenesis of S. aureus but SB has been
recognized for its importance in severe disease
phenotypes and promotion of staphylococcal
virulence in abscess and endocarditis models of
infection (13, 14). Additionally, the SB
biosynthetic locus and sirABC are amongst the
most strongly up-regulated genes in bacteria
isolated from the iron-restricted host (15–17).
Importantly, SB biosynthesis can occur
independent of TCA cycle activity, which is
down-regulated in S. aureus as part of the iron-
sparing response (18, 19). In contrast, SA
biosynthesis is dependent on the TCA cycle for
citrate precursors (18).
ketoglutarate (α-KG) and citrate, and two
molecules of L-2,3-diaminopropionate (L-Dap)
(21). These essential precursor molecules are
synthesized by SbnA, SbnB, and SbnG. SbnG is a
dedicated citrate synthase that produces citrate
from oxaloacetate and acetyl-CoA (18, 23, 24). α-
KG and L-Dap are provided by the sequential
enzymatic activities of SbnA and SbnB which use
substrates O-phospho-L-serine (OPS) and L-
glutamate (25, 26). OPS is postulated to be
shunted from the serine biosynthesis pathway
stemming from 3-phosphoglycerate produced in
glycolysis (Fig. 1) (18, 26).
SbnI, the ninth gene product, is a heme-
responsive transcriptional regulator for SB
production (27). SbnI is required for full
expression of sbnD-H and thus controls SB-
mediated iron acquisition. SbnI binds a 271-bp
DNA fragment in the sbnC coding region.
However, SbnI can bind heme which abrogates
interaction with DNA thereby limiting SB
production. A model is proposed by which SbnI is
required for transcription of the full SB
biosynthetic operon and senses heme to reduce
SB synthesis in favor of heme acquisition (27).
This model also provides a mechanism for the
findings that S. aureus demonstrates a heme-iron
preference in vitro (28).
Based on primary sequence analysis,
homology was not detected between SbnI and any
characterized transcription factors or heme-
binding proteins (27), but it is annotated as
containing an N-terminal ParB-like domain. ParB
is an essential component of the chromosome
segregation system in bacteria (29). However, the
conserved N-terminal ParB-like domain in SbnI is
not responsible for interaction with DNA in ParB
(30), leaving the role of the ParB-like domain in
SbnI unknown.
Staphyloferrin biosynthesis is achieved
through Non-ribosomal peptide synthetase-
Independent Siderophore (NIS) biosynthesis (20,
21). NIS synthesis systems involve intermediates
and enzymes that are freely dissociable from each
other during assembly. Primary metabolites
generally serve as the source of building blocks to
generate siderophores and can be first chemically
modified prior to incorporation into the
siderophore. In addition to the synthetases
required for assembly of the siderophore, NIS
biosynthetic gene clusters generally encode
enzymes that synthesize the necessary precursors
to be assembled by the NIS synthetases (22).
The aim of this study was to gain insight
into how SbnI functions using X-ray
crystallography. The SbnI structure revealed
striking structural homology to a recently
characterized free serine kinase, SerK, from the
archaea T. kodakarensis (31). SerK can
phosphorylate free L-serine using ADP to
generate OPS for cysteine biosynthesis (32).
Herein, we demonstrate that SbnI is also a free
serine kinase that uses ATP to phosphorylate L-
serine to yield OPS and ADP (Fig. 1). The
structure of SbnI, supported by site-directed
The SB biosynthetic operon consists of
nine genes, sbnABCDEFGHI. SbnC, SbnE, and
SbnF are the three synthetases and SbnH is a
decarboxylase that together are required for SB
assembly from one molecule of each α-
2

SbnI serine kinase activity and role in SB biosynthesis
mutagenesis, suggests that it follows a similar
open-close reaction mechanism as proposed for
SerK. Additionally, SbnI-generated OPS can be
used by SbnA in vitro and serves as the in vivo
source of OPS for SB production. To our
knowledge, this is the first example of a bacterial
free serine kinase and the first described free
serine kinase that is ATP dependent. This
enzymatic function earns SbnI an enzymatic role
in the SB biosynthetic pathway in addition to its
2A) and contains an absolutely conserved GXXR
59-62
motif, GVHR
in SbnI.
Domain II is composed of a mixed a/b
fold with a central 4-stranded antiparallel b-sheet
surrounded by 5 a-helices. A pair of antiparallel
b-strands abut the main sheet and serve as a linker
to Domain I. B-factor analysis reveals that
domain II has a relatively high average B-factor
2
2
of 109 Å , compared to 75 Å in domain I,
suggesting domain II has more disorder in the
crystal and the domains are connected by a
flexible linker.
heme-dependent
function.
transcriptional
regulatory
To gain functional insight into SbnI, a
Results
1-240
search of the SbnI
structure against structures
Structure determination of SbnI
in the PDB was performed with the Dali server.
Five proteins all belonging to the functionally
diverse superfamily of ParB/Srx proteins were
identified (Z score < 3.9). The most striking
observation was the high structural similarity SbnI
Full length (254 amino acid) SbnI was not
amendable for X-ray crystallography due to its
propensity to precipitate. To improve solubility
and stability in solution, several expression
constructs were made containing varying N- and
C-terminal truncations based on predicted
secondary structure and disorder identified using
PSIPRED and DISOPRED, respectively (33, 34).
One construct containing a 14-amino acid C-
terminal truncation, SbnI , had improved
stability in solution and produced well-diffracting
crystals. The structure of selenomethionine-
labelled SbnI
resolution using single wavelength anomalous
dispersion in space group P3 with one molecule
shared with the top search result, SerK (PDB ID:
1-240
5
(
X0B). Superimposition of SbnI
with SerK
PDB ID: 5X0B) using PDBeFOLD has a root
mean square distance (RMSD) of 2.0 Å for 194
Cα despite sharing only 19% amino acid sequence
identity across the aligned residues (Fig. 2B). The
other structures also share modest sequence
identity (12-23%) and include sulfiredoxin (Srx)
from Homo sapiens (PDB ID: 2RII, RMSD of 2.7
Å across 84 residues), chromosome partitioning
protein (ParB) from Sulfolobus solfactaricus
1
-240
1
-240
was determined to 2.5 Å
1
in the asymmetric unit (Fig. 2A). Data collection
and refinement statistics are summarized in Table
(
PDB ID: 5K5D, RMSD of 2.5 Å across 71
residues), oncogenic suppressor (Osa) from
Shigella flexneri (PDB ID: 4OVB, RMSD of 3.7
1. All 240 residues were modeled with 98% of
residues in the most favored regions of the
Ramachandran plot.
Å
across 83 residues), and chromosome
segregation protein (Spo0J) from Thermus
thermophilus (PDB ID: 1VZ0, RMSD of 5.4 Å
across 76 residues) (31, 36–39).
1
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SbnI
is comprised of two domains,
domain I and domain II. Domain I consists of
residues M1-Q83 and I205-A240 and domain II is
composed of residues Y84-N204. Domain I
SerK is a free serine kinase from T.
kodakarensis that uses ADP to phosphorylate L-
serine to generate OPS for cysteine biosynthesis.
Of the proteins annotated in the ParB/Srx family,
SerK is the only identified kinase though Osa and
Srx both possess ATPase activity (36, 40).
Overall, the structures of SerK and SbnI are very
includes
a
conserved core ParB/Srx fold,
corresponding to the annotated ParB-like domain
based on primary sequence. This fold has been
described in a functionally diverse ParB/Srx
superfamily of proteins. Members are found in
varied biological contexts and thus far are
described to bind a nucleotide for kinase, ATPase,
or DNase activity (35, 36). To our knowledge, no
ParB/Srx family member has been found to be
directly involved in siderophore biosynthesis. The
ParB/Srx core domain is comprised of a 4-strand
mixed β-sheet and two α-helices, a2 and a3 (Fig.
similar. The SerK domain II has a high average B-
1-240
factor and superimposition of SbnI
with the
SerK structure in a “closed” conformation (PDB
ID: 5X0E) suggests how SbnI may possess
similar domain flexibility in solution (Fig. 2C).
Additionally, structural superimposition and
3

SbnI serine kinase activity and role in SB biosynthesis
multiple sequence alignments revealed that
several residues important for substrate and
product binding identified in the SerK crystal
structure (PDB ID: 5X0E) are conserved in SbnI.
Moreover, the active site architecture is highly
conserved between the two proteins (Fig. 2D, Fig.
S1). Of the active site residues, SerK Glu30 was
identified as a catalytically essential residue and
Asp69 is required for magnesium ion binding;
site-directed mutagenesis of either of these
residues abolished SerK kinase activity (31). The
homologous residues in SbnI are Glu20 and
Asp58.
groove between domain I and II (Fig. 4AB).
Structural alignment with the structure of the
SerK ternary product complex (PDB ID: 5X0E)
revealed that among SbnI homologs, the putative
active site is highly conserved, while the
remainder of the protein surface is variable (Fig.
4C).
SbnI is a dimer in solution
The oligomeric state of SbnI was
analyzed using dynamic light scattering (DLS).
Since SbnI contains 7 Cys residues, the analysis
was conducted in the presence of GSH as a
reductant. The calculated molecular weight based
on amino acid sequence of full-length SbnI is 30
kDa and the measured molecular weight by DLS
was 61 ± 6 kDa with an average of 24 ± 7% of
polydispersity, implying it predominantly forms a
dimer in solution (Fig. S2). The molecular weight
The genomic context of sbnI was
analysed to compare with the genomic
neighborhoods of homologs. All staphylococcal
sbnI homologs are part of the nine gene SB
biosynthetic cluster. More distant homologs co-
occur with sbnA and sbnB homologs, either alone
or in combination with different putative
siderophore biosynthetic enzymes (Fig. 3). More
distant SbnI homologs are shorter and alignments
suggests that they have an abbreviated domain II
1
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of SbnI
by DLS was 28 ± 3 kDa with 34 ± 1%
1
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polydispersity (Fig. S2). SbnI
has a calculated
weight of 28 kDa, implying it is primarily a
monomer in solution. These data indicate that the
C-terminal 14 amino acids excluded from the
(Fig. S1). Interestingly, a putative sbnI ortholog
1
-240
was identified upstream of the sbnA-H gene locus
in Ralstonia solanacearum, which was previously
thought to lack a SbnI homolog but still produces
SB (41). A multiple sequence alignment of SerK
and SbnI homologs used in the genomic
neighborhood analysis reveals that certain key
residues important for catalysis, substrate, and
product binding identified in SerK are fully
conserved (Fig. S1). Notably, these include the
SerK catalytic residue Glu30, magnesium ion
binding residue Asp69, residues implicated in
interacting with the β phosphate of ADP or
phosphate group of OPS, His72 and Arg73, and
residues that interact with the serine moiety of
OPS, Trp102 and Thr223 (Fig. S1). More
variability is seen with the SerK residues
interacting with the adenosine group, raising the
possibility that SbnI and other homologs may use
a different phosphate donor or binding-mode.
Overall, the genomic neighborhood and sequence
analyses suggest that free serine kinases are found
in diverse species belonging to Firmicute and
Proteobacteria phyla.
SbnI
construct are important for dimerization
of the full-length protein.
SbnI is a serine kinase that uses L-serine and
ATP to generate OPS
OPS is a substrate for SbnA in SB
biosynthesis (25, 26), lending support to our
hypothesis that SbnI produces OPS for use by
SbnA. To thus test if SbnI is capable of producing
OPS, the spectral changes that occur when SbnA
binds OPS were used to assay SbnI activity. SbnA
has a characteristic absorption maxima at 412 nm
attributed to an internal Schiff base formed
between its pyridoxal 5’-phosphate (PLP)
cofactor and an active site lysine. Adding OPS to
SbnA causes a rapid change in UV-Vis spectra
with the appearance of absorption peaks at 324
nm and 467 nm (Fig. 5A), characteristic of the
formation of an external aminoacrylate
intermediate (25). This spectral change is specific
to OPS and does not occur with O-acetyl-L-serine
or L-serine (25). SbnA incubated with SbnI, L-
serine, and ADP resulted in no change in the UV-
Vis spectrum of SbnA. However, SbnA incubated
with SbnI, L-serine, and ATP led to a shift in the
UV-Vis spectra indicative of OPS production and
reaction with SbnA-PLP to form the external
1
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Consurf analysis of SbnI
map conserved regions to the molecular surface
Fig. 4). Highly conserved residues including
those that form the kinase active site delineate a
was used to
(
4

SbnI serine kinase activity and role in SB biosynthesis
aminoacrylate (Fig. 5BC). No spectral change was
observed when SbnI was omitted indicating that
only the SbnI enzymatic product could react with
SbnA-PLP. Therefore, we conclude that the
reaction product is most likely OPS and SbnI
activity is ATP-dependent.
Phosphate acceptors, alternative to L-
serine, were tested using a pyruvate kinase/lactate
dehydrogenase (PK/LDH) assay for detection of
ATP conversion to ADP. L-threonine, α-KG, and
L-Dap were not phosphate acceptors (data not
shown). Additionally, SbnI did not phosphorylate
the serine residue in a His-Ser dipeptide (data not
shown).
together, these data allow us to conclude that
these mutants are catalytically inactive. These
data also correlate with SbnA-PLP UV-Vis
absorption spectra that demonstrated that these
SbnI variants also do not produce OPS (Fig.
5DE). The importance of these residues in
catalysis is consistent with the reaction
mechanism presented for SerK in which substrate
binding promotes conformational closure
positioning the catalytic Glu30 (Glu20 in SbnI)
close to the hydroxyl group of bound L-serine.
Glu30 is a catalytic base that deprotonates the
hydroxyl group of L-serine. The deprotonated
hydroxyl can then attack the phosphorus atom of
the ADP b-phosphate to yield OPS and AMP
(31). Our results suggest SbnI uses a similar two-
ligand binding sequential mechanism, but instead
uses L-serine and ATP to yield ADP and OPS.
SbnI-mediated conversion of ATP to
ADP and generation of OPS were monitored
3
1
using HPLC and P NMR. Incubation of SbnI
with ATP and excess L-serine led to turnover of
ATP to ADP as detected by HPLC (Fig. 6A)
indicating SbnI has ATPase activity. Unlike SerK,
no turnover of ADP to AMP was detected with
ADP as a phosphate donor by HPLC (Fig. 6A).
ADP was not generated with when L-serine was
excluded, indicating that this activity requires the
Kinetic analysis of SbnI kinase activity
To obtain kinetic parameters for the SbnI
kinase activity, SbnI enzymatic turnover was
monitored using an established coupled assay for
ADP using PK/LDH. The steady-state kinetic
parameters of SbnI reaction with ATP and L-
serine were determined and are summarized in
Table 2. The saturating concentration of L-serine
was beyond conditions permissive to the assays
3
1
presence of L-serine. P NMR was also used to
monitor SbnI-mediated ATPase activity and
generation of OPS from L-serine. The observed
31
disappearance of the ATP g-phosphate P signal
31
with the concomitant appearance of a P signal of
OPS demonstrates transfer of the ATP g-
phosphate to L-serine to yield ADP and OPS (Fig.
and thus K could not be accurately determined.
m
A second assay to measure SbnI enzymatic
turnover employed SbnA-dependent turnover of
OPS coupled to a phosphate release detection
assay. SbnA is the most likely in vivo acceptor of
OPS generated by SbnI to facilitate synthesis of
SB substrates L-Dap and a-KG, in concert with
SbnB. Using excess concentrations of SbnA, the
coupled assay demonstrated that SbnA could use
SbnI-generated OPS and supplied L-glutamate to
generate its products, N-(1-amino-1-carboxyl-2-
ethyl)-glutamic acid and inorganic phosphate. The
kinetic parameters of SbnI reaction with ATP and
L-serine using the SbnA coupled assay were
determined and are summarized in Table 2. The
kinetic parameters measured using both methods
7
). The chemical shift of SbnI-generated OPS was
3
1
consistent with the P NMR spectrum measured
for an OPS standard (Fig. S3). Attempts to obtain
a co-crystal structure of SbnI with identified
substrates
(or
ATP
analog,
adenyl-
imidodiphosphate) or products, both in the
presence and absence of heme, have not been met
with success.
SbnI active site variants
The role of Glu20 and Asp58 in SbnI
kinase function were tested by site-directed
mutagenesis. Two mutants each containing a
single alanine substitution, SbnI E20A and SbnI
D58A, were generated. Using the PK/LDH assay,
we determined that the mutants were incapable of
turning over ATP to ADP in the presence of L-
serine. Additionally, no ADP could be detected by
HPLC in reactions containing the SbnI mutants
incubated with ATP and L-serine (Fig. 6B);
agree with rates and K values in the same order
m
of magnitude. SbnI mutants E20A and D58A
were catalytically insufficient to accurately
measure enzyme rates. Compared to SerK, SbnI
has a K for ATP one order of magnitude lower
m
than SerK has for ADP and a higher K for L-
m
serine by 2-orders of magnitude.
5

SbnI serine kinase activity and role in SB biosynthesis
1
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The enzymatic activity of SbnI
was
To test if SbnI could serve as a source of
OPS for SB biosynthesis, the serC transposon
insertion mutant was tested for its ability to make
SB. A disc diffusion assay was used to detect SB
in spent culture supernatant of serC and sbnI
transposon insertion mutants, compared to the
wildtype USA300 S. aureus strain. Briefly, spent
culture supernatants were applied to sterile filter
discs on iron-restricted agar seeded with either
sirA or htsABC mutant S. aureus strains. The sirA
mutant strain is defective for SB uptake so growth
promotion around the disc in iron-restricted media
is dependent on the presence of SA in the
supplied culture supernatant. The htsABC mutant
strain is unable to take up SA and thus growth
around the disc is reliant on the presence of SB in
supplied culture supernatant. Similar to wildtype
S. aureus, the serC transposon insertion mutant
was capable of producing SB (Fig. 8B), indicating
the presence of another pathway for OPS
production independent of SerC, almost assuredly
via SbnI (Fig. 1).
also measured since the 14-amino acid C-terminal
truncation does not exclude any regions in the
SerK structures identified as required for substrate
and product binding or catalysis. SbnI
displayed decreased kinase activity compared to
full-length SbnI. These data correlate with the
1
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1
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HPLC and spectral data with SbnA for SbnI
having intermediate activity relative to full-length
SbnI (Fig. 5F, 6B). Plots of initial velocities used
for determination of kinetic constants are included
in the supporting information (Fig. S4). Given
1
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that SbnI
is a monomer in solution (see
above), we conclude that dimerization/
oligomerization of SbnI is not essential for its
kinase activity.
Physiological function of ATP-dependent serine
kinase activity in S. aureus
The serC gene encodes the enzyme
responsible for OPS synthesis from 3-
phosphohydroxypyruvate in the S. aureus serine
biosynthesis pathway. To our knowledge, prior to
this work, SerC activity was the only identified
metabolic source of OPS in S. aureus and was the
assumed source of OPS for SB biosynthesis (18,
The sbnI mutant was impaired for SB
production (Fig. 8B), consistent with previously
published results using the disc diffusion assay
(27). Decreased SB production by the sbnI mutant
is due to the necessity of SbnI for full expression
of sbnDEFGH (27). A serC sbnI double mutant
is expected to be SB deficient. However, as the
sbnI mutation is pleiotropic on SB biosynthesis
due to its requirement for transcription of sbn
biosynthetic genes, assaying for SB production
does not inform on a role in OPS production.
Nonetheless, our results support the hypothesis
that SbnI serves as a second metabolic route for
OPS biosynthesis from L-serine in S. aureus.
Production of OPS from L-serine and ATP is a
previously unrecognized route for OPS synthesis
in bacteria.
2
6). To test if other metabolic sources of OPS
exist in S. aureus, wildtype USA300 and a serC
transposon insertion mutant USA300 strain (serC)
were grown in chemically defined media
containing glucose with and without L-serine in a
TECAN plate reader. In L-serine replete medium,
both wildtype USA300 and the serC mutant grew,
although the serC mutant entered stationary phase
at a lower cell density and failed to reach
equivalent biomass as that of the WT culture (Fig.
8
A). However, in stark contrast, in a growth
medium lacking L-serine, wildtype USA300 grew
while the serC mutant failed to grow altogether
(Fig. 8A). Thus, we conclude that the serC
transposon mutant is a bona fide serine auxotroph;
a serine auxotrophy phenotype was previously
observed for an Escherichia coli serC mutant
strain (42). We also conclude that SerC activity is
the sole source of OPS for serine biosynthesis;
since the medium is iron-restricted, SbnI would be
expressed and, if it contributed to OPS
biosynthesis for use in the L-serine biosynthetic
pathway, we would have not observed L-serine
auxotrophy for the serC mutant (see Fig. 1).
Discussion
SbnI is a free L-serine kinase that makes
OPS, which serves as a substrate for SbnA and is
a precursor for SB biosynthesis. Insight into the
kinase function of SbnI was gained based on
homology identified by structural similarity with
SerK, an enzyme in a biosynthetic pathway for
cysteine in the thermophilic archaea T.
kodakarensis (31). ATP is used as the phosphate
donor in the reaction catalysed by SbnI, whereas
SerK uses ADP. Interestingly, hyperthermophiles
6

SbnI serine kinase activity and role in SB biosynthesis
generally use ADP in place of ATP in key
glycolytic enzymes as an adaptation to life at high
temperatures, presumably because ADP is more
stable than ATP (43). While SbnI is capable of
phosphorylation of free L-serine, it was not
capable of mediating phosphotransfer to serine
residues within a His-Ser dipeptide. Bacterial
Ser/Thr kinases such as S. aureus Stk1 (also
named PknB) (44) and cognate phosphatases
function as molecular switches that have key roles
in bacterial cell signaling as in eukaryotic systems
heterotrophic mechanism of amino acid
metabolism (35). Interestingly, this cysteine
synthase is a distant SbnA homolog and a true
OPS sulfhydrylase. Also, SerK is postulated to
provide an advantage by enabling carbon from
serine to be directed to glycolysis and
gluconeogenesis by conversion to OPS (35). In
vitro evidence suggests that S. aureus uses amino
acids to support gluconeogenesis (46). However,
serine is used to generate ATP and acetate rather
than to facilitate gluconeogenesis as in T.
kodakarensis (35, 46). In contrast, SbnI kinase
activity fulfils a distinct physiological role,
providing substrate necessary for SB production.
(45). However, SbnI free L-serine kinase activity
is functionally and enzymatically distinct from
that of Ser/Thr kinases.
1
-240
SbnI
retained partial catalytic
The comparatively low kcat/K
m
may allow SbnI to
1
-
efficiency compared to full-length SbnI. As SbnI
respond to greater range of substrate
a
2
40
is lacking 14 residues at the C-terminus and is
concentrations, such that at high L-serine
concentrations SbnI increases the rate of OPS
production for SB production and possibly other
metabolic processes. Though SbnI has lower
catalytic efficiency than SerK (Table 2), the
related ParB/Srx family member, Srx, is similar
to SbnI as it is not a highly efficient enzyme. The
Srx ATP-dependent reduction of peroxidreoxin
sulfinic acid activities described in mammals and
plants so far are slow, with turnover rates of 0.2-
monomeric, either the C-terminus or dimerization
is required for full kinase function. The residues
shown to be required for phosphotransfer in SerK
(Glu30, Asp69) are conserved in SbnI and present
in the truncated protein. Moreover, three of four
residues interacting with the serine substrate in
SerK are conserved (Glu30, Trp102, Thr223) and
the fourth position is a conservative substitution
of His225 with Phe203 in SbnI (Fig. 2D). In
contrast, interactions of SerK with ADP/AMP are
poorly conserved. Of eight residues making key
contacts, only three residues (Ser43, His72,
Arg73) are conserved in SbnI and two of these
interact with the phosphate groups. Attempts to
obtain crystals with substrates or products bound
-
1
0.5 min for ATP, which are an order of
magnitude lower than SbnI (47–49). However, the
-1
-
catalytic efficiencies of Srx are 0.8-8.4 mM min
1
and in the range measured for SbnI (47, 49).
SbnI is a sufficient biological source of
OPS for SB biosynthesis and contributes to the
functional modularity of the sbn locus. A
recognized characteristic of bacterial networks is
1
-240
to SbnI or SbnI
have not been met with
success and the binding mode of ATP to SbnI
remains elusive.
a
high degree of modularity and sparse
Structural analysis of SerK suggests that
the conformational closure upon binding both
substrates positions the catalytic glutamate
connectivity between individual functional
modules, where a functional module refers to a
group of biological components that are spatially
isolated or chemically specific and work together
for a discrete biological function (50). The
functional redundancy of enzymes encoded in the
sbn locus for generation of precursor substrates,
SbnG and SbnI, decreases dependence on central
metabolism and contribute to the modularity of
the sbn locus. SbnG is functionally redundant
with the TCA cycle citrate synthase, CitZ (23).
Moreover, SbnG activity allows for SB
biosynthesis to occur independent of TCA cycle
activity (18). This functional independence is
important because S. aureus elicits an iron-
sparing response during infection resulting in
(Glu30) to deprotonate L-serine to attack the
terminal phosphate of ADP. With no published
structure of substrate or product-free SerK, the
1
-240
apo-SbnI
structure supports this proposed
mechanism in that the unbound form is in an open
conformation to expose the binding pocket.
Measurement of SbnI enzyme kinetic parameters
revealed that, compared to SerK, it has a
relatively low selectivity for L-serine. The
comparatively low kcat/K
m
could relate to the
physiological role of SbnI in S. aureus. SerK
supplies cysteine synthase with OPS to produce
cysteine and may represent an ancient
7

SbnI serine kinase activity and role in SB biosynthesis
down-regulation of the TCA cycle. We propose
that the functional redundancy of SbnI with SerC
for OPS production allows SB biosynthesis to
occur independent of glycolysis as SerC substrate
innovation to support adaptation to a more
invasive lifestyle. In contrast, SA biosynthetic
genes are found across both coagulase-positive
staphylococci and more commonly commensal
coagulase-negative staphylococci.
is
funneled
from
3-phosphoglycerate.
Additionally, the serine biosynthetic pathway is
regulated by negative feedback where SerA,
metabolically upstream of SerC, is allosterically
inhibited by L-serine and could limit the amount
of SerC-derived OPS available to support SB
synthesis when serine is abundant. Thus, an
alternative, SB-dedicated, OPS synthetic route via
SbnI is advantageous. Together with SbnG, SbnI
allows SB biosynthesis to occur autonomously
from glycolysis and TCA cycle activity by
generating precursor substrates dedicated to SB
biosynthesis.
This newly determined enzymatic role of
SbnI generates questions regarding how it carries
out heme-dependent transcriptional regulation of
the sbn locus. It remains unclear if SbnI kinase
activity
impacts
its
heme-dependent
transcriptional regulation of the sbn locus. The
1
-240
structure of SbnI
did not reveal a prototypical
DNA-binding motif or heme binding site. The
functional sites required for kinase activity could
be structurally distinct from those involved in the
transcriptional regulation and heme-binding. Full-
1-240
length SbnI is a dimer yet SbnI
is a monomer
This observed metabolic redundancy may
in the crystal structure and in solution. The C-
1
-
improve robustness and help buffer environmental
perturbations S. aureus encounters during
infection. Glucose is the preferred carbon source
by S. aureus and available at concentrations to
support growth in human blood (51). However,
within staphylococcal abscesses, glucose is
limiting and S. aureus likely survives through
catabolism of secondary carbon sources,
specifically lactate, peptides, and free amino acids
terminal 14 amino acids excluded from the SbnI
2
40
construct
are
likely
required
for
multimerization, which could be important for
transcriptional regulatory function or heme-
binding. Additionally, SbnI has a C-terminal
extension of 34 amino acids compared to SerK
and this C-terminal region is truncated in distant
SbnI homologs not associated with a SB
biosynthetic locus. This raises the possibility that
kinase activity may be the core function of the
non-SB-associated SbnI homologs. The C-
terminal extension in SbnI could be a structural
adaptation to facilitate multiple biological
functions in SB precursor biosynthesis and sbn
locus gene regulation. Heme-dependent SbnI
regulatory activity may further enhance network
robustness as a negative feedback loop from an
iron acquisition perspective. Characterization of
SbnI heme-binding and effect on kinase activity
remains the subject of future work.
(
46, 52, 53). Resultant changes in central
metabolism of S. aureus may not severely hinder
SB biosynthesis due to the modularity of the sbn
system. Despite variability in growth conditions,
one constancy in S. aureus survival strategy is
iron acquisition.
Serine is amongst the most abundant
amino acids found in fluid obtained from S.
aureus infected prosthetic joints. SB biosynthetic
genes including sbnI, but not SA biosynthetic
genes, are up-regulated in this environment (17).
Also, L-serine is one of the most rapidly
consumed amino acids by S. aureus grown on
amino acids in vitro (46). Together, the
combination of high L-serine in the extracellular
milieu and the rapid consumption of L-serine may
limit the activity of the endogenous serine
biosynthetic pathway. Under these conditions
OPS from serine biosynthesis is likely to be
limiting to feed SB biosynthesis justifying the
necessity of SbnI-generated OPS. Interestingly,
SB production is restricted to more invasive
coagulase-positive staphylococcal strains and the
sbn locus may represent a lineage-specific
OPS production by the kinase activity of
SbnI represents a new biosynthetic path for
production of this metabolite in bacteria. SbnI
may serve as a metabolic adaptation to facilitate
SB production when growing on non-preferred
carbon sources. Furthermore, it demonstrates the
metabolic flexibility S. aureus possesses to allow
employment of iron uptake strategies in changing
host environments.
Experimental procedures
Cloning and site-directed mutagenesis
8

SbnI serine kinase activity and role in SB biosynthesis
Constructs with an N-terminal His
6
tag
ratio by weight of His
6
protein to remove the His
6
and thrombin cleavage site were generated in
pET28a vectors for recombinant expression of S.
aureus full-length SbnI, residues 1-254, with the
first codon mutated from the native TTG to a
common start codon, ATG, and the S. aureus C-
tag over 18 hr at 4°C. Subsequently, recombinant
protein was dialysed into 50 mM HEPES (pH
7.4), 5% (v/v) glycerol, and 2 mM TCEP and
further
purified
by
anion
exchange
chromatography using a Source 15Q column (GE
Healthcare). Purified protein was obtained by
elution with a NaCl gradient and further dialyzed
into 50 mM HEPES (pH 7.4), 300 mM NaCl, 5%
(v/v) glycerol, 2 mM GSH. The sample was
concentrated to approximately 20 mg/mL, flash
frozen, and stored at -80°C. Selenomethionine-
1
-240
terminal truncated construct SbnI
(residues 1-
240). The S. aureus SbnI nucleotide sequence can
be accessed in the GenBank database under
accession code NC_009641.1 (90178-90942)
(gene locus NWMN_RS00380) and the amino
acid sequence can be accessed through NCBI
Protein Database under NCBI accession
WP_001015549.1. Briefly, a megaprimer-based
whole-plasmid synthesis PCR cloning protocol
was used to clone constructs amplified from
chromosomal DNA from S. aureus strain
Newman (54). S. aureus SbnI variants E20A and
D58A were produced using a single primer
mutagenesis method (55). Primers used in this
study are summarized in Table S1. All clones
were introduced into E. coli BL21 (λDE3) and
confirmed by DNA sequencing. Bacterial strains
and plasmids used in this study are summarized in
Table 3.
1
-240
incorporated SbnI
was produced by methods
previously described (56) and purified as
1
-240
described above for native SbnI
.
His -tagged SbnA was expressed in E.
6
coli BL21 (λDE3) cells from the plasmid pET28a,
purified by His-tag affinity chromatography, and
digested with the thrombin to remove the His tag.
6
The protein was further purified by anion
exchange chromatography using the previously
published method for improved SbnA solubility
(26). SbnA was dialyzed into 50 mM Tris pH 8,
100 mM NaCl, and 2 mM TCEP, concentrated to
approximately 20 mg/mL and stored at -80°C
Protein expression and purification
Crystallization, data collection, and structure
1-240
1-240
Recombinant full-length SbnI, SbnI
,
determination of SbnI
1
-240
SbnI E20A, and SbnI D58A constructs were
overexpressed in E. coli BL21 (λDE3) cells.
Cultures were grown in 2xYT media
supplemented with 25 µg/mL kanamycin at 30°C
to an OD600 of 0.7-0.9. Cultures were then
induced with 0.5 mM isopropyl β-D-
thiogalactopyranoside and grown for an additional
Selenomethionine-labeled
SbnI
crystals were grown by sitting drop vapor
diffusion at 4°C in 2 uL drops with a 1:1 mixture
1
-240
of ~20 mg/mL SbnI
in 50 mM HEPES (pH
7.4), 100 mM NaCl, 5% (v/v) glycerol, and 2 mM
TCEP with reservoir solution containing 0.18 M
HEPES (pH 7.5) and 20% (w/v) PEG 8000.
Crystals were briefly soaked in reservoir buffer
supplemented with 30% (v/v) glycerol for
cryoprotection and flash frozen in liquid nitrogen.
A single wavelength anomalous diffraction
dataset was collected at the Canadian Light
Source on Beamline 08B1-1 (57). The data was
processed and scaled using XDS (58, 59).
18
h
at 20°C. Cells were pelleted by
centrifugation at 4400 x g for 7 min at 4 °C and
resuspended in buffer containing 50 mM HEPES
(pH 7.4), 300 mM NaCl, 5% (v/v) glycerol, 2 mM
tris(2-carboxyethyl)phosphine (TCEP), and 10
mM imidazole on ice. Approximately 5 mg of
DNase was added to cell suspension prior to lysis
at 4°C using an EmulsiFlex-C5 homogenizer
Crystals were of space group P3
1
with one
(
Avestin). Insoluble material was removed by
molecule in the asymmetric unit. Five
selenomethionine sites were identified for phasing
to build a preliminary model using AutoSol
(initial figure of merit of 0.35) and Autobuild
(187 of 240 residues built) programs in Phenix
(60). Manual building was done using Coot (61)
and refinement was performed with phenix.refine
using translation liberation screw parameters with
centrifugation at 39,000 x g for 1 h and
recombinant protein was purified from soluble
lysate using a HisTrap nickel affinity column (GE
Healthcare) by elution with an imidazole gradient.
Protein was dialyzed against 50 mM HEPES (pH
7
.4), 100 mM NaCl, 5% (v/v) glycerol, and 2 mM
TCEP and then cleaved with thrombin at a 1:500
9

SbnI serine kinase activity and role in SB biosynthesis
three groups (62). The refined structure contains
binds OPS was used to evaluate OPS production
by SbnI. The spectrum of 15 µM SbnA in 50 mM
HEPES pH 7.4, 100 mM NaCl, 5% (v/v) glycerol,
1
240
Met -Ala , one glycerol, and 12 water
molecules. Data collection and refinement
statistics are summarized in Table 1. Structure
figures were generated in PyMOL (The PyMOL
Molecular Graphics System, Version 1.8
Schrödinger, LLC). Domain analysis was done
using the Dali server for comparison of the
protein structure against structures in the PDB
20 mM MgCl
was recorded before and after the addition of 15
2
, 25 mM L-serine, and 5 mM ATP
1-240
µM SbnI, SbnI E20A, SbnI D58A, or SbnI
.
Phosphate donor specificity was also examined
using 5 mM ADP in place of ATP.
(63).
HPLC
Kinase activity of SbnI was detected
using HPLC to examine production of ADP from
ATP. The reaction mixture was composed of 50
mM HEPES (pH 7.4), 100 mM NaCl, 2.5%
glycerol, 50 mM L-serine, 0.25 mM ATP or ADP,
Genomic neighborhood and conservation
analysis
Protein homology between genomic
regions carrying SbnI orthologs was plotted using
a custom Biopython script bio.links.py based on
output from BLASTP 2.2.2.28 (e-value ≤ 1.00e-
10 mM MgCl , and 5 µM of SbnI, SbnI E20A, or
2
SbnI D58A. The reaction was carried out for 1
hour at room temperature (22ºC). The protein was
removed by centrifugation using a 3K nanosep
column. The filtrate was 0.2 µm filtered and
analyzed by HPLC using a Waters 2695
Separations HPLC module (Milford, MA)
equipped with a Waters 2996 photodiode array
detector and a Luna 3 µm PFP(2) 50 x 4.6 mm LC
column (Phenomenex) using a linear gradient of 0
to 15% methanol in 0.1 M ammonium acetate, pH
40)
(https://github.com/minevskiy/bioinformatics).
Species used for comparison were found by
BLAST search of SbnA or SbnI and STRING
analysis of SbnI. Orthologous genes are indicated
in the same color.
Sequence conservation was mapped onto
1
-240
the SbnI
structure using ConSurf (64). The
multiple sequence alignment used for the analysis
was generated using default ConSurf parameters
and the SbnI amino acid sequence as the search
sequence.
-
1
4.5 over 10 minutes at 1 mL min . Analytes were
detected by the absorbance at 258 nm.
3
1
P NMR spectra of SbnI kinase reaction
Dynamic light scattering
The reaction mixture contained 50 mM
1-240
Samples of SbnI and SbnI
analyzed by DLS using a DynaPro Plate Reader
Wyatt Technologies). Protein was diluted to 0.5
were
HEPES (pH 7.4), 100 mM NaCl, 2.5% (v/v)
glycerol, 10 mM MgCl , 48 mM L-serine, 5 mM
ATP, and 5% D O. NMR spectra were collected
2
(
2
mg/mL with 50 mM HEPES (pH 7.4), 300 mM
NaCl, 5% (v/v) glycerol, and 2 mM GSH and
results were generated based on averaging five, 5
second acquisitions. Data were collected at room
temperature. Values reported are an average of
data collected.
at 25 °C using a broadband frequency probe with
Z-magnetic field gradient in a Bruker Avance III
3
1
500 MHz spectrometer. One-dimensional
P
spectra were recorded at different time points
before and after addition of 4.8 µM SbnI until
reaction completion. The spectra were referenced
to 2,2,6,6-tetramethylpiperidine, which was set to
0 ppm. The spectra were processed and using
TopSpin™ (Bruker). The chemical shifts of ATP
(65) and OPS were assigned using reference
spectra (Fig. S3).
UV-Vis Spectrophotometry analysis of SbnI OPS
production using SbnA
UV-Vis spectra were collected using
Varian Cary 50 UV-Vis spectrophotometer.
SbnA-PLP spectrum was recorded at
concentration of 15 µM in 50 mM HEPES pH 7.4,
00 mM NaCl, 5% (v/v) glycerol. The spectrum
a
Measurement of serine kinase activity
1
ATP-dependent serine kinase activity of
of SbnA aminoacrylate aldimide complex was
recorded immediately after addition of 30 µM
OPS (25). The spectral shift observed when SbnA
SbnI was measured using
kinase/lactate dehydrogenase (PK/LDH) coupled
assay. The assay is based on a reaction in which
a
pyruvate
1
0

SbnI serine kinase activity and role in SB biosynthesis
the regeneration of hydrolyzed ATP is coupled to
the oxidation of NADH (66). The rate of NADH
assay was run for 15 minutes. To determine
kinetic parameters, the initial velocities of SbnI
kinase reactions in the presence of varying
concentrations of ATP with 100 mM L-serine and
in the presence of varying concentrations of L-
serine with 10 mM ATP were recorded. All data
was collected on a Varian Cary 50 UV-Vis
spectrophotometer at room temperature (22ºC)
and a total of three replicates were collected for
each reaction condition. The concentration of
inorganic phosphate release from OPS was
determined using the extinction coefficient A360 nm
-
absorbance decrease at 340 nm (A340 nm = 6220 M
1
-1
cm ) is proportional to the rate of ATP
conversion to ADP by SbnI-kinase activity.
Coupled reactions contained 50 mM HEPES pH
7
.4, 100 mM NaCl, 2.5% (v/v) glycerol, 10 mM
MgCl , 2 mM phosphoenolpyruvate, 1/50 of the
final reaction mixture volume of PK/LDH enzyme
from rabbit muscle, Sigma-Aldrich, cat. P-0294),
mM ATP, and 100 mM L-serine. The mixture
2
(
5
was incubated for 5 min to remove any
contaminating ADP. Continuous measurement at
-1
-1
= 11000 M cm (67). Data were fit by nonlinear
regression using a Michaelis-Menten model in
GraphPad Prism 6.
3
40 nm was recorded for 2 minutes prior to
1
-240
addition of 0.5 µM SbnI, SbnI , SbnI E20A, or
SbnI D58A enzyme to start the reaction. The
assay was run for 10 minutes. To determine
kinetic parameters, the initial velocities of SbnI
S. aureus bacterial strains and growth
conditions
1
-240
and SbnI
kinase reactions in the presence of
Experiments were performed with a
derivative of S. aureus USA300 LAC cured of the
27-kb plasmid encoding macrolide resistance
(68). The plasmid-cured USA300 LAC is referred
to as USA300 throughout. Transposon insertion
varying concentrations of ATP with 100 mM L-
serine and in the presence of varying
concentrations of L-serine with 5 mM ATP were
recorded. Phosphate donor specificity was
investigated with 10 mM ADP and phosphate
acceptor specificity was examined with 50 mM L-
threonine, and 50 mM His-Ser dipeptide. In
addition, alternative substrates, 50 mM L-Dap and
R
mutants JE2 serC::ΦNΣ; Em (SAUSA300_1669)
R
and JE2 sbnI::ΦNΣ; Em (SAUSA300_0126)
were obtained from the Nebraska Transposon
Mutant Library (NTML) containing the resistance
cassette ermB, which confers resistance to
erythromycin (69). Transposons were transduced
to USA300 background strain using phage 80α
and confirmed by PCR using published methods
50 mM a-KG were tested as phosphate acceptors.
All data was collected on a Varian Cary 50 UV-
Vis spectrophotometer at room temperature
(22ºC) and a total of three replicates were
collected for each reaction condition.
(70). USA300 serC::ΦNΣ and
USA300
SbnI kinase activity was also measured
sbnI::ΦNΣ transposon insertion mutants are
referred to as serC and sbnI throughout (Table 3).
Bacterial growth curves to test serine auxotrophy
were performed in chemically-defined medium
with 0.4% (w/v) glucose (CDMG) as previously
described (18), with and without L-serine. Briefly,
colonies of wildtype USA300 or serC transposon
insertion mutant were inoculated from tryptic soy
agar into 2 mL CDMG overnight at 37ºC. Cells
were normalized to an OD600 of 0.1 and washed
twice with CDMG lacking L-serine (CDMG-Ser)
and 5 µL of the resuspension was used to
inoculate 200 µL aliquots of CDMG or CDMG-
Ser in 96 well plates. Cultures were grown in
TECAN plate reader for 24 hours at 37ºC with 10
second shake every 10 min and the OD600 was
assessed every 30 min. Data are representative of
three independent experiments, and error bars
signify standard error of the mean.
using an assay with SbnA. In a reaction mixture,
SbnI-dependent OPS production was measured
using SbnA and L-glutamate to release inorganic
phosphate and N-(1-amino-1-carboxyl-2-ethyl)-
glutamic acid (26). A coupled enzymatic assay
was used to detect inorganic phosphate release
from OPS as previously described (67). The
reaction mixture contained 50 mM HEPES pH
7
.4, 100 mM NaCl, 2.5% (v/v) glycerol, 10 mM
MgCl , 10 mM ATP, 100 mM L-serine, 0.2 U
2
purine nucleoside phosphorylase, 400 µM 2-
amino-6-mercapto-7-methylpurine riboside, 5 µM
SbnA, 2 mM L-glutamate. The mixture was
incubated for 10 min to remove any
contaminating inorganic phosphate and establish a
baseline. Continuous measurement at 360 nm was
recorded for 2 minutes prior to addition of 0.5 µM
1
-240
SbnI or SbnI
enzyme to start the reaction. The
1
1

SbnI serine kinase activity and role in SB biosynthesis
ddH O. To assess growth promotion of
2
Disc diffusion assays to assess siderophore
production
concentrated culture supernatants, S. aureus strain
RN6390 sirA mutant (growth of this mutant is
dependent on SA in supernatant) or htsABC
mutant (growth of this mutant is dependent on SB
in supernatant) derivatives, as previously
described (72), were seeded into TMS agar
containing 10 µM ethylenediamine-N, N’-bis(2-
Concentrated spent culture supernatants
were prepared from 10 mL cultures of S. aureus
USA300, serC transposon insertion mutant, and
sbnI transposon insertion mutant grown for 16 hrs
in Chelex-100 treated Tris minimal succinate
5
(TMS), as previously described (71), in a flask to
hydroxyphenylacetic acid) to 2 x 10 cells/mL. 10
volume ratio of 10:1 at 37ºC with shaking at 200
rpm without antibiotic selection. Growth was
assessed via OD600 and culture densities
normalized. Bacterial cells were pelleted by
centrifugation and culture supernatants were filter
sterilized and lyophilized overnight. Dried
material was resuspended in 0.5 mL sterile
µL of concentrated supernatant was applied to
sterile paper discs placed on TMS agar containing
the seeded reporter strains, and growth radii about
the discs were measured after 24 hr incubation at
37ºC. The reported growth radius has the disc
radius (3 mm) subtracted. Statistical analyses
conducted
using
2-way
ANOVA.
Acknowledgments:
We thank Kateryna Ievdokymenko for assistance with genomic neighborhood analysis and figure
generation. We also thank Dr. Anson Chan for assistance with data collection and Mariko Ikehata and
Angele Arrieta for technical assistance. We thank the staff at the CLS for assistance with crystallographic
data collection.
This work was supported by the Canadian Institutes of Health Research (CIHR) grants to M.E.P.M
(MOP-49597) and D.E.H. (MOP-38002). M.M.V. is supported by a PGSD scholarship from the Natural
Sciences and Engineering Research Council. Support for infrastructure for structural biology was
provided by the Canadian Foundation for Innovation to M.E.P.M. Research described in this paper was
performed using beamlines 08B1-1 and 08ID-1 at the Canadian Light Source, which is supported by the
NSERC, the National Research Council Canada, the Canadian Institutes of Health Research, the Province
of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.
1
-240
The atomic coordinates for the crystal structure of SbnI
is available in the Research Collaboratory for
Structural Bioinformatics Protein Databank under PDB # 5UJE.
Conflict of interest:
The authors declare that they have no conflicts of interest with the contents of this article.
1
2

SbnI serine kinase activity and role in SB biosynthesis
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7
FOOTNOTES
The abbreviations used are: SA, staphyloferrin A; SB, staphyloferrin B; NIS, non-ribosomal peptide
synthetase-independent siderophore; α-KG, α-ketoglutarate; L-Dap, L-2,3-diaminopropionate; OPS, O-
phospho-L-serine; PLP, pyridoxal 5’-phosphate; PK/LDH, pyruvate kinase/lactate dehydrogenase;
RMSD, root mean square deviation; DLS, dynamic light scattering; TCEP, tris(2-
carboxyethyl)phosphine; CDMG, carbon defined medium with glucose; TMS, Tris minimal succinate.
1
6

SbnI serine kinase activity and role in SB biosynthesis
Tables
1
-240
Table 1. Data collection and refinement statistics for SbnI
.
Data Collectiona
Resolution Range (Å)
Space group
42 – 2.50 (2.59 – 2.50)
P3
1
Unit cell dimensions
a, b, c (Å)
Unique reflections
Completeness (%)
Redundancy
55.1, 55.1, 92.7
10,883 (1,564)
99.9 (100)
2.9 (2.9)
Average I/σI
15.8 (2.2)
0.051 (1.007)
56.4
R
merge
2
Wilson B-factor (Å )
Anisotropy
0.501
Refinement
R
work (Rfree
)
0.219 (0.259)
12
0.003
Number of water molecules
r.m.s.d. bond length (Å)
Average B-values (Å )
2
95.0
Ramachandran plot (%)
Most favored regions
Disallowed regions
97.5
0.4
PDB ID
5UJE
a
Data collection values in parentheses represent the data for the highest resolution shell.
1
-240
Table 2. Steady-state kinetic parameters of SbnI and SbnI
.
-1
-1
-1
K
m
(mM)
k
cat (min )
k
cat/K (mM min )
m
ATP
SbnI
0.6 ± 0.1
0.2 ± 0.1
1.2 ± 0.3
3.9 ± 0.1
3.9 ± 0.1
2.1 ± 0.1
6.8 ± 1.0
17.3 ± 3.6
1.7 ± 0.7
SbnIa
SbnI1-240
ADP
SerKb
L-serine
SbnI
SbnIa
SbnI1-240
2.4 ± 0.5
12240 ± 720
5100
340 ± 40
150 ± 20
900 ± 450
5.1 ± 0.5
14.3 ± 0.8
10.0 ± 0.6
2.1 ± 0.1
0.04 ± 0.01
0.07 ± 0.01
0.02 ± 0.01
2600
SerKb
13100 ± 300
a
Kinetics measured using SbnA coupled assay
Data from Makino et. al. (32)
b
1
7

SbnI serine kinase activity and role in SB biosynthesis
Table 3. Bacterial strains and plasmids used in this study.
Bacterial strains and
plasmids
Description
Source or
reference
Strains
E. coli
BL21 (λDE3)
F− ompT gal dcm lon hsdS
gene 1 ind1 sam7 nin5])
(r
B
−
m
B
+)λ (DE3 [lacI lacUV5-T7 Novagen
B
S. aureus
USA300
USA300 LAC cured of antibiotic resistance plasmid
(68)
(69)
(69)
This study
This study
(73)
R
JE2 serC::ΦNΣ
JE2 sbnI::ΦNΣ
serC
sbnI
sirA
JE2 serC::ΦNΣ; Em (SAUSA300_1669)
R
JE2 sbnI::ΦNΣ; Em (SAUSA300_0126)
R
USA300 serC::ΦNΣ; Em (SAUSA300_1669)
R
USA300 sbnI::ΦNΣ; Em (SAUSA300_0126)
R
RN6390ΔsirA::Km ; SB transport-deficient mutant
R
htsABC
RN6390ΔhtsABC:Tc ; SA transport-deficient mutant
(72)
Plasmids
pET28a-sbnI
pET28a-sbnI1
pET28a-sbnI-E20A
pET28a-sbnI-D58A
IPTG-inducible expression vector containing sbnI; KmR
IPTG-inducible expression vector containing sbnI1-240; KmR
IPTG-inducible expression vector containing sbnIE20A; Km
This study
This study
This study
This study
-240
R
R
IPTG-inducible expression vector containing sbnID58A; Km
a
R
R
R
Em , Km , and Tc designate resistance to erythromycin, kanamycin, and tetracycline respectively.
Figures
Figure 1. Metabolic pathways for the production of L-Dap and a-KG from glucose or L-serine in S.
aureus. Highlighted in light green is the contribution of SbnI in this pathway, which feeds into the
previously characterized SB biosynthetic pathway (light blue). 3P stands for 3-phospho.
1
8

SbnI serine kinase activity and role in SB biosynthesis
1
-240
1-240
Figure 2. Structure of SbnI . The overall fold of SbnI
is shown as a cartoon and divided into two
domains with N- and C-termini and alpha helices labelled. Domain I shown in teal contains the
conserved, core ParB/Srx domain (a2 and a3) and domain II is colored raspberry. (B) Structural
1
-240
superimposition of SbnI
C) Superimposition with the “closed” conformation of SerK (blue, PDB 5X0E) in cartoon putty
representation, where the size of the tube depends on the B-factor in each structure. (D) Structural
(raspberry), and the “open” conformation of SerK (yellow, PDB ID: 5X0B).
(
1
-240
superimposition of SaSbnI
residues, AMP, and OPS are drawn as sticks. Mg is drawn as a green sphere, O, N, and P atoms colored
red, blue, and orange, respectively. AMP and OPS carbons are colored cyan.
(raspberry), and SerK (yellow, PDB ID: 5X0E). Selected active site
2
+
1
9

SbnI serine kinase activity and role in SB biosynthesis
Figure 3. Illustration of gene neighborhoods containing SbnI homologs from diverse species from
Firmicute and Proteobacteria phyla. Each predicted gene is represented by an arrow showing the direction
of transcription. Grey links connect protein homologous with e-value≤1e-40 and orthologous genes are
indicated in the same color. This figure highlights that SbnI homologs appear in the same genomic
context as SbnA and SbnB homologs in these bacterial genomes. The bottom scale shows the length of
depicted genomic regions in nucleotide base pairs. Region coordinates used for each species are as
follows (GenBank:nucleotide region): Staphylococcus aureus USA300 FPR3757 (CP000255.1:134324-
1
45881), Staphylococcus pseudintermedius E104 (LAWU01000001.1:151604-163186), Brevibacillus
badius NBRC 110488 (NZ_BDFB01000004.1:355053-396579), Paenibacillus larvae SAG 10367
NZ_CP020557.1:4429364-4439754), Bacillus badius DSM 5610 (NZ_LVTO01000018.1:5060-15111),
Marininema mesophilum DSM 45610 (NZ_FNNQ01000001.1:200002-206967), Methylobacterium
nodulans ORS 2060(NC_011894.1:7032971-7044380), Ralstonia solacearum CQPS-1
NZ_CP016915.1:169574-186973), and Shewanella denitrificans OS217 (NC_007954.1:663307-
74451).
(
(
6
2
0

SbnI serine kinase activity and role in SB biosynthesis
1
-240
Figure 4. Conservation of surface residues of SbnI
generated using ConSurf. (A, B) Ribbon and
1
-240
surface representations of SbnI have AMP and OPS modelled based on a structural alignment with the
SerK ternary product complex (PDB ID: 5X0E). (C) Surface representation of SbnI
1
-240
after 180° rotation
about the x-axis. Conserved amino acids are colored maroon, residues of average conservation are white,
and variable amino acids are turquoise.
Figure 5. Detection of OPS by SbnI via reaction with SbnA. (A) UV-visible absorption spectra of SbnA
and SbnA in complex with OPS. (B) Spectra of SbnA incubated with SbnI-generated OPS from L-serine
and ATP but not (C) ADP. SbnI variants (D) E20A and (E) D58A are defective in producing OPS as
1
-240
indicated by no change in the SbnA absorption spectra. (F) SbnI
slower rate compared to full-length SbnI.
is capable of producing OPS but at a
2
1

SbnI serine kinase activity and role in SB biosynthesis
Figure 6. HPLC analysis of the nucleotide reaction products. HPLC trace of ATP, ADP, and AMP
standards (black lines) with retention times 1.4, 1.5, and 2.9 min, respectively. (A) SaSbnI, L-serine, and
ADP (green line) or ATP (blue line), control reactions of ATP and L-serine excluding SbnI (red line) and
1
-240
of SbnI and ATP excluding L-serine (grey line). (B) Reactions of variants SaSbnI
D58A (red line), or SbnI E20A (blue line) with ATP and L-serine.
(green line), SbnI
3
1
Figure 7. A stack plot of P NMR spectra for a single reaction of SbnI mediated conversion of L-serine
and ATP to OPS and ADP. The reaction was initiated by the addition of SbnI with a final concentration
of 4.8 µM. The initial concentrations of ATP and L-serine were 5 and 48 mM, respectively. The reaction
buffer contained 50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM MgCl , and 2.5% (v/v) glycerol.
2
2
2

SbnI serine kinase activity and role in SB biosynthesis
Figure 8. (A) Growth kinetics of S. aureus USA300 wildtype (black lines) and serC transposon insertion
mutant (red lines) strains in chemically defined medium with glucose (CDMG) (solid lines) and CDMG
without L-serine (CDMG – L-Ser) (dashed lines). (B) Agar plate disc diffusion bioassays were performed
using culture supernatants prepared from S. aureus USA300 strains (wildtype and sbnI and serC
transposon insertion mutants), as indicated on the x-axis, that were grown for 16 hrs in chelex-treated Tris
minimal succinate (c-TMS) media. The black dots [labeled SA (DsirA)] are a measure for the presence of
SA in culture supernatants and the grey dots [labeled SB (DhtsABC)] are a measure of SB in culture
supernatants based on the growth radius around the disc. The disc radius (3 mm) is subtracted from the
reported growth radius. Lines represent the standard deviation from the mean. *** p-value < 0.0002, ****
p-value < 0.0001.
2
3

SbnI is a free serine kinase that generates O-phospho-L-serine for staphyloferrin B
biosynthesis in Staphylococcus aureus
Meghan M. Verstraete, Cecilia Perez-Borrajero, Kirstin L. Brown, David E. Heinrichs and
Michael E. P. Murphy
J. Biol. Chem. published online February 26, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA118.001875
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