Structural and functional characterization of the phosphoglucomutase from Xanthomonas citri subsp. citri

Article abstract of DOI:10.1016/j.bbapap.2016.08.014

Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named “pathogenicity, virulence and adaptation”. One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.

Full text of DOI:10.1016/j.bbapap.2016.08.014

Structural and functional characterization of the phosphoglucomutase from
Xanthomonas citri subsp. citri
Leandro Seiji Goto, Andr e´ Vessoni Alexandrino, Camila Malvessi Pereira,
Carla Silva Martins, Humberto D’Muniz Pereira, Jos e´ Brand a˜ o-Neto, Maria
Teresa Marques Novo-Mansur
PII:
DOI:
Reference:
S1570-9639(16)30170-4
doi: 10.1016/j.bbapap.2016.08.014
BBAPAP 39805
To appear in:
BBA - Proteins and Proteomics
Received date:
Revised date:
Accepted date:
25 May 2016
17 August 2016
23 August 2016
Please cite this article as: Leandro Seiji Goto, Andr ´e Vessoni Alexandrino, Camila
Malvessi Pereira, Carla Silva Martins, Humberto D’Muniz Pereira, Jos ´e Brand ˜a o-Neto,
Maria Teresa Marques Novo-Mansur, Structural and functional characterization of the
phosphoglucomutase from Xanthomonas citri subsp. citri, BBA - Proteins and Proteomics
(2016), doi: 10.1016/j.bbapap.2016.08.014
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ACCEPTED MANUSCRIPT
Title: Structural and functional characterization of the phosphoglucomutase from
Xanthomonas citri subsp. citri
*1
1
1
Authors: Leandro Seiji Goto , André Vessoni Alexandrino , Camila Malvessi Pereira ,
1
2
3
Carla Silva Martins , Humberto D'Muniz Pereira , José Brandão-Neto , Maria Teresa
1
Marques Novo-Mansur
1
Affiliations: Laboratório de Bioquímica e Biologia Molecular Aplicada - LBBMA,
Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos,
2
SP, Brazil; Laboratório de Biologia Estrutural, Grupo de Cristalografia, Instituto de Física
3
de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil, Diamond Light Source,
Harwell Science and Inovation Campus, Didcot, UK
*
Correspondence to: Leandro S. Goto - Laboratório de Bioquímica e Biologia Molecular
Aplicada - LBBMA, Departamento de Genética e Evolução, Universidade Federal de São
Carlos, São Carlos, SP; Rod. Washington Luiz, km 235, Monjolinho, ZIP code 13565-905,
PO Box 676, SP, Brazil; Phone/Fax: +55 (16) 3351-8788; E-mail: seiji_l@hotmail.com
1

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ABSTRACT
Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all
economically important varieties of citrus. Studying Xanthomonas genes related to the
invasive capacity may improve the knowledge on how this works and ultimately use the
information to avoid the disease. Some annotated genes from Xanthomonas citri subsp.
citri published genome are addressed to an interesting class of genes named "pathogenicity,
virulence and adaptation". One of them is xanA, which encodes a predicted
phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living
kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion
of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to
synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a
characterization of this gene product is presented by structural and functional studies.
Molecular cloning was used for heterologous expression and deletion of xanA. A
Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein
structure was also determined by X-ray diffraction on the recombinant enzyme substrate-
free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was
done with a suicide plasmid construct and the obtained mutant was tested for pathogenic
capacity. This study is the first describing the properties of the Xanthomonas citri subsp.
citri phosphoglucomutase.
KEYWORDS
Citrus canker; Xanthomonas citri; phosphoglucomutase; protein structure and function;
gene deletion
Abbreviations: ASU, asymmetric unit; MPD, Methyl-2,4-pentanediol; PGM, Phosphoglucomutase.
2

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1
- INTRODUCTION
Citrus canker caused by Xanthomonas citri subsp. citri is one of the most
devastating diseases on citrus crops. It affects all commercial varieties and can easily
spread on citrus growing areas worldwide. The disease causes local brownish corky lesions
in all aerial parts of the plant, including leaf and fruit. Infected fruit loses quality due to
lesions and productivity due to general premature fruit fall. Pathogen transmission is done
by contact, either by natural agents like winds, rain, insects, or by human handling. As a
major concern, there is no effective treatment nor prevention to the disease and the
workaround is usually done by eradicating affected plants and groves [1].
Efforts made to better understand the mechanisms of infection include genomic [2-
4
], transcriptomic [5] and proteomic [6-8] studies as ways to indicate possible key
molecules or mechanisms which could be used to control the disease. Random [9-11] and
targeted [12-14] mutations are among the approaches commonly used on gene-to-disease
correlation studies and the annotated genome [2] may provide initial perspectives on the
selection of target genes. X. citri subsp. citri genomic records [2] describe a "pathogenicity,
virulence and adaptation" gene class, which includes the gene xanA (XAC3579) encoding a
predicted phosphoglucomutase (PGM). Our recent proteomics studies showed that X. citri
subsp. citri PGM have positive differential expression from plain LB medium to
pathogenicity-inducing medium (XAM-M), possibly indicating some involvement with the
disease (Artier et al., personal communication).
Phosphoglucomutases belong to the -D-phosphohexomutase enzyme superfamily,
widespread in all organisms. All enzymes in this superfamily catalyze the reversible
conversion of 1- and 6-phosphosugars differing in the specific hexose [15] although some
3

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PGMs may have bifunctionality, acting on both glucose and mannose [16-17]. All of them
share general structural and functional features including a highly conserved phosphoserine
residue in the active site that participates in the phosphoryl transfer, requirement of a
divalent metal ion for activity and the same reaction mechanism. In brief, a
monophosphoryl sugar is transiently converted into a biphosphorylated intermediate that is
dephosphorylated and released as a product [18]. Catalyzing the conversion of glucose-1-
phosphate (G1P) to glucose-6-phosphate (G6P), PGMs are enzymes (EC 5.4.2.2) that play
roles in carbohydrate metabolism. G6P may efficiently follow the Glycolytic or Pentose
Phosphate pathways, differently from G1P, the reverse reaction product which generally is
involved in the glycogen metabolism [19-20].
Beyond the possible functions in energetic resources mobilization, a notable
function of PGM in Xanthomonas comes from its relationship with pathogenicity, due to
involvement in the biosynthesis of xanthan, an important interface in pathogen-plant
interaction [21-22]. Activity of the X. citri subsp. citri PGM should provide 1-
phophosugars for some precursors of xanthan [23-24].
In this work, molecular cloning was used to characterize the xanA (XAC3579)
product, the predicted X. citri subsp. citri PGM, by functional and structural studies on the
recombinant protein (XcPGM) and by infection studies with the deleted mutant.
2
2
- MATERIALS AND METHODS
.1 - General procedures - Xanthomonas citri subsp. citri 306 was stored at -80° C in 20%
v/v glycerol stocks. Custom oligonucleotides were provided by IDT. Restriction enzymes,
PCR reagents and cloning vectors were supplied by Fermentas. pNPTS138 was kindly
supplied by Prof. Dr. Ferreira H. (UNESP, Rio Claro-SP, Brazil). Other reagents were of
4

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analytical grade or higher. Molecular biology techniques used throughout this work were
described [25], or are detailed as follows.
2
.2 - Cloning and construction of expression and deletion systems - Isolation of DNA
fragments of interest was done by PCR using X. citri subsp. citri 306 genomic DNA as
template. For recombinant expression purposes, PGM encoding sequence was amplified
using oligonucleotides designed to incorporate an NdeI restriction site (underlined) at the 5’
end of the PCR product (5’CCTTCATATGCCCATGACGCTACCCGCCTTC) and an
XhoI site at the 3’ extremity (5'TAGTATACTCGAGTCAGCCGCGCAGCAGGTTAG),
right after XAC3579 predicted stop codon. For gene deletion, two distinct 1 kb regions
adjacent to the PGM encoding sequence were individually amplified. For the upstream
region, HindIII and BamHI restriction sites were adapted to the 5'
(5'TATATAAGCTTTTCCGACTGCAGCCACACATCGACG)
and
3'
(5'GTACTAGGATCCATCCTGAAGTGGGGGACGCC) ends respectively. For the
downstream
region,
BamHI
(5'TATATCTAGGATCCTTGACCCCTCTCCCACCCATAGAC)
and
NheI
(5'
CAATAGCTAGCACCACATCCTCAAGCCCCACC) sites. PCR was performed using a
C1000 Touch (Biorad) thermal cycler programmed to execute a 97° C initial 7 min
denaturing step, followed by 35 cycles of 94° C for 30 s, 60° C 30 s, 72° C 1.5 min, and a
final elongation step of 10 min at 72° C. Each fragment was amplified using 100 ng of
genomic DNA and 100 pMol of each oligonucleotide on defined pair combinations in 50 μl
reactions. Amplification products were gel-purified, cloned into pJET 1.2 (Fermentas) and
transformed into E. coli DH5α for propagation. All the plasmids inserts were completely
confirmed by sequencing [26] in a 3130 Genetic Analyzer (Applyed Biosystems). DNA
5

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encoding PGM was excised using NdeI and XhoI restriction enzymes and subcloned into
pET28a (Novagen) respective sites. The constructed plasmid provides IPTG induced
expression of XcPGM fused to an N-terminal His-tag. The 1 kb regions were excised from
the cloning vectors using their respective adapted restriction sites (HindIII-BamHI and
BamHI-NheI) and sequentially subcloned into pNPTS138 (Alley Dickon, unpublished
R
results) respective sites. The constructed deletion vector carries a kan selection marker and
also provides sucrose suicide selection [27].
2
.3 - Recombinant expression and protein purification – The expression vector was
transformed into E. coli BL21 (DE3) (Novagen) and expression was carried out in an
orbital shaker at 250 rpm, 18° C in LB broth added of 30 μg/mL kanamycin for 16 h after
0
.1 mM IPTG addition to culture in mid-log growing stage. Cells from 1 L culture were
collected by centrifugation and resuspended in 50 mL of 25 mM Tris-HCl pH 8.0 100 mM
NaCl. Cell lysis was done by ultrasound pulses under ice bath and insoluble cellular debris
were removed by centrifugation. Lysate soluble extract was loaded onto a 5 mL Ni-NTA
column (Novagen) pre-equilibrated with the same buffer. For IMAC purification, column
with bound protein was washed with 50 mL of 2 mM imidazole on the same buffer and
purified XcPGM was eluted in 5 column volumes of 200 mM imidazole on the same
buffer. Imidazole was removed by dialysis against 25 mM Tris-HCl pH 8.0 100 mM NaCl
and XcPGM was stored at -20° C. Aliquots from every XcPGM preparation were routinely
reserved for SDS-PAGE analysis [28] (not shown). Protein for crystallization was
concentrated by ultrafiltration (Centriprep, Millipore) in 12.5 mM Tris-HCl pH 8.0 50 mM
NaCl. XAC3579 encodes a putative 450 amino acid residues protein with molecular mass
6

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of 49.1396 kDa. Measurement of PGM concentration was done by absorbance at 280 nm
-1
using a calculated [29] extinction coefficient of 0.1% (g.L ) = 0.842.
2
.4 - Enzyme activity assay - PGM activity was measured [30] using Phosphoglucomutase
Activity Assay Kit ab155896 (Abcam). Samples were prepared according to manufacturer
instructions, except for the amount of substrate G1P which was adjusted for kinetics assays.
Briefly, the assay infers G6P production by a glucose-6-phosphate dehydrogenase coupled
+
reduction of NAD . NADH then reacts with a probe that begins to absorb light at 450 nm.
Absorbance is converted to NADH equivalents by a standard curve, corresponding to G6P
amount. Measurements were done in an iMark (Biorad) microplate reader, using clear flat
bottom 96 well microtiter plates, recording the absorbance at 450 nm every 7 s along 20
min at 25° C. Initial enzyme velocities were estimated as the maximum linear rates of
absorbance increase at 450 nm, using 0.0041 mg/mL XcPGM samples in 100 L reactions
(0.0834 M). Absorbance values were correlated to product equivalent amounts with the
standard curve equation Abs450 nm = 0.0052 + 0.0822 x [NADH] and initial velocities were
4
determined as a function of substrate concentrations. Substrate was varied from 4 x 10 to
1
1
9.53 M G1P replacing water volume with a serial dilution of G1P, to a total volume of
00 L. PGM activity was also tested on cell lysates aliquots of 50 L from wild type X.
citri subsp. citri and the xanA deleted mutant (Xac∆3579), also adjusted to a final volume
of 100 L.
2
.5 - Protein crystallization and structure determination – XcPGM was prepared as
described (section 2.3) and concentrated to 11 mg/mL in 12.5 mM Tris-HCl pH 8.0, 50 mM
NaCl. Crystallization attempts were done using a Honeybee 939 liquid handler (Genomic
Solution) with several available crystallization screens, by the vapor diffusion method.
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Suitable XcPGM substrate-free crystals were obtained in condition A8 of Morpheus kit
(Molecular Dimensions), which consists of 12.5 % PEG 1000, 12.5 % PEG 3350, 12.5 %
MPD, 30 mM MgCl , 30 mM CaCl and 100 mM MOPS/HEPES pH 7.5. Crystals
2
2
appeared after 3 days and took a week to reach their maximum size (around 0.3 mm in the
longest dimension). The substrate-free crystal was cooled in liquid nitrogen and diffraction
data was obtained up to 1.27 Å resolution on beamline I24 at Diamond Light Source in the
UK. The Xia2 software [31] was used for data processing. XcPGM structure was solved by
molecular replacement using Phaser [32] and the structure of phosphoglucomutase from
Pseudomonas aeruginosa (PDB ID 2FKF) [33], which shares 34% sequence identity, as a
search model. The resultant solution was submitted to Arp/wArp [34] for automatic model
building and the structure was refined using both Phenix [35] and Coot [36].
XcPGM ligand-bound structures were obtained using crystals from condition A4 of
Morpheus kit (12.5 % PEG 1000, 12.5 % PEG 3350, 12.5 % MPD, 30 mM MgCl , 30 mM
2
CaCl , 100 mM MES/Imidazole pH 6.5), after a 24 hours soaking with 2 mM of G1P or
2
G1,6P. Then, crystals were diffracted in a Rigaku MicroMax 007 HF with R-AXIS IV++
(Rigaku Co.), using copper radiation at Laboratório de Biologia Estrutural, IFSC-USP.
These data were processed with XDS Package [37]. XcPGM ligand-bound structures were
also solved by molecular replacement using Phaser and the previously refined XcPGM
ligand-free structure. Ligand sugars were positioned with Coot and refinements were
performed using Phenix [35-36]. The behavior of R and Rfree were used as main criteria for
validating the refinement and stereochemical quality of the models evaluated with
Molprobity [38]. Statistics of data processing and refinement are shown in Table 1.
8

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The coordinates and structure factors for PGM substrate-free, G1P bound and G1,6P
bound structures are deposited in the PDB (PDB IDs: 5BMN, 5BMP, 5KL0, respectively).
2
.6 - Gene deletion and studies with the mutant (Xac∆3579) - As described before
(section 2.2), two 1 kb flanking regions of the xanA structural gene were subcloned in
tandem into pNPTS138. One g of this constructed vector was used to transform
electrocompetent cells of wild type X. citri subsp. citri strain 306 using a 0.2 cm gap
cuvette in a Gene Pulser Xcell (Biorad) and parameters were 2.5 kV pulse, 200 Ω resistance
and 25 µF capacitance. Transformants were selected on LB agar added of kanamycin. To
obtain plasmid-free double crossing-over deleted mutants from these, an isolated
transformant was grown for 24 h at 30° C, 250 rpm in LB broth without antibiotics. After
that, 0.2 L of the culture were spread over LB agar added of 10 % sucrose. As the
constructed vector carries sacB suicide mark, growing colonies must lack the plasmid and
should comprise both unchanged cells and the expected deleted mutant. Screening was
done by PCR using another pair of oligonucleotides specific for X. citri subsp. citri
genomic DNA, flanking 50 bp before the 1 kb upstream region and 50 bp after the 1 kb
downstream region. Thus, deleted mutants are expected to show a PCR product around 2 kb
while wild type should be close to 3.4 kb (see supplementary material).
Mutant and wild type cells were cultivated for 48 h at 30° C and 250 rpm in LB
broth for both artificial infection of Citrus aurantifolia leaves [9] and native PGM activity
tests. Cells were collected by centrifugation, washed twice with sterile water and
resuspended to OD600 nm = 0.59. Detached Citrus aurantifolia leaves were separately
inoculated by spreading 10 L of these bacterial suspensions over homogeneously pin
9

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punctured areas on the abaxial surfaces and placed inside sterile clear tubes with 3 mL of
sterile water. Negative control was done with sterile water. The development of infections
was followed visually for 15 days at 25° C (see supplementary material). The remaining
bacterial suspensions were added to 1 mM Tris-HCl pH 8.0 4 mM NaCl and lysed with
ultrasound pulses. The lysates supernatants were used on the PGM enzymatic assay
described before. Additionally, general colony morphology was monitored on plain LB
agar and pathogeny inducing XAM-M medium, a derivative from XAM-1 [39] proposed by
Prof. Dr. Ferro from Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal,
Universidade Estadual Paulista, UNESP-Jaboticabal, São Paulo, Brazil (unpublished).
3
3
- RESULTS
.1 - Enzyme activity
Heterologous expression provided around 25 mg of purified XcPGM from 1 L of
+
LB medium. XcPGM activity was estimated using an NAD reduction probe coupled to
G6P oxidation. Using G1P as substrate, PGM produces G6P, which is oxidized by glucose-
+
6
-phosphate dehydrogenase into 6-Phosphogluconolactone using NAD as electron
+
acceptor. NAD is reduced to NADH which reacts with a probe that begins to absorb light
at 450 nm. Hence, absorbance corresponds to G6P production and initial XcPGM reaction
rates are recorded varying the amount of substrate G1P. Data is presented as a substrate
saturation curve fitted in the Michaelis-Menten model (Figure 1). XcPGM has apparent
Vmax of 4.04 mol/min/mg of protein and a K of 0.65 mM using G1P as substrate.
m
3
.2 - Protein structure
XcPGM crystallized in orthorhombic space group P2 2 2 with one molecule per
1
1 1
ASU and the crystals diffracted at high resolution (1.27-1.85Å) (Table 1). The general
1
0

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structure (Figure 2A) is very similar to other PGMs, a globular protein with the four
phosphohexomutase alpha/beta/alpha typical domains.
When compared by superposition, the ligand-free structures of XcPGM and P.
aeruginosa PGM (PDB ID 1K35) showed an RMSd of 1.95Å. Major differences were
observed in domain IV, especially in the orientation of helices 16 and 18 (residues 400-411
and 454-469 respectively), and beta-sheets were more conserved. Other subtle difference is
observed in the turn between helices 5 and 6 of domain II.
The three XcPGM structures described here are quite similar to each other (Figure
2
0
B), with an RMSd of 0.45Å (G1,6P-bound structure compared with ligand-free) and
.131Å (G1P-bound with G1,6P-bound). The protein bound with either G1P or G1,6P
shows a small decrease in both b and c cell dimensions (Table 1) to accommodate a small
rotation in the domain IV. The major difference between ligand-free and bound structures is
observed in the conformation of the second and third strands of the domain IV and the β-
turn comprised by them (431-450), which is in "opened" conformation in ligand-free
structure and in "closed" conformation in either G1P and G1,6P bound structures (Figure
2
B). Indeed, seven (G1P) or eight (G1,6P) hydrogen bonds are formed between the ligand
and the residues 431-450 (not shown). These hydrogen bonds can be responsible for the
conformational change of this region upon ligand interaction. The conformational change is
more pronounced in the β-turn region comprised by residues 436-445 and the greatest
change is made by the C of T440, which moves 6.5 Å upon G1,6P binding.
3
.3 - Structural characterization of the active site
In the XcPGM active site, both ligands G1P and G1,6P were readily identified in
the standard Fo-Fc electron density maps contoured at 3 (Figure 3A and B). Both ligands
1
1

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induced several structural changes that can be used to characterize the XcPGM active site.
The beta-hairpin where a conserved phosphoserine 119 lies changes its conformation upon
ligand binding and the side chains of residues R34 and K129 adopt different rotamers. R34
in the ligand-free structure anchors two water molecules which are displaced upon ligand
binding. Also, R34 side chain interacts with the D32 side chain (D32 OD1 - R34 NE, D32
OD2 - R34 NE and D32 OD2 - R34 NH2) until a ligand molecule arrives. Then, R34 side
chain changes conformation opening the active site. The presence of the ligand also induces
a conformational change in the main chain of the region 323-326 (between the third strand
of the β-sheet 4 and the helix 11, and also in the side chain of H325). In the ligand-free
structure, the side chain of H325 interacts with Y348 (H325 ND1 - Y348 OH). In ligand-
bound structures, the region 323-326 moves toward the ligands and interactions of the main
chain of H325 and the ligand are observed (H325 N - O3 (G1,6P) 2.8Å, N325 N - O4
(G1P) 3.1Å). Simultaneously, the interaction between H325 side chain and Y348 is lost.
Also, there are minor displacements of side chains of R302, S344 and H346 induced by
ligand binding.
In G1P binding, 15 hydrogen bonds are formed (2 water-mediated) in the active site
(Figure 4A). Binding of the G1,6P involves the formation of 21 hydrogen bonds with the
active site surroundings, six of them via water molecules (Figure 4B). One of these water
molecules (w200 in G1,6P structure) is absent in the G1P complex, due to the sugar
positioning. However, this structured water could possibly interact with glucose 6-
phosphate (G6P), although only in the predictions as we did not obtain the XcPGM-G6P
complex.
3
.4 - Studies with the mutant
1
2

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Two types of colonies were observed after plasmidial suicide selection on sucrose
0 %: a normal looking variety and a smaller, clearer, less viscous type (Figure 5). PCR
1
screening confirmed that they were indeed the wild type and the expected deleted mutant
respectively. Deleted mutants (Xac∆3579) showed a PCR product close to 2 kb whereas
unchanged cells included the PGM 1.4 encoding region, showing a 3.4 kb product. Also,
western blotting analysis confirmed lack of PGM (not shown). Further corroborating xanA
deletion, Xac∆3579 lysates did not show PGM activity, different from the wild type (Figure
6
1
). As an additional data, our tests indicated that X. citri subsp. citri should have around 5 x
-12
0
PGM U/cell in the growth conditions we had used.
Xac∆3579 took 3 days to develop visible colonies on agar LB and 16 days on
pathogenicity-inducing agar XAM-M, contrasting with the wild type that takes 2 and 3
days respectively. This indicates that although not being essential, PGM should have an
important role on X. citri subsp. citri growing in XAM-M pathogenicity-inducing
environment [39]. This correlates well with our local studies which indicated that PGM is
differentially expressed from one medium to the other (Artier et al., personal
communication). However, in vivo experiments can provide better suited environment for
correlation of PGM activity with the canker disease. Thus, detached Citrus aurantifolia
leaves were simultaneously infected with either the mutant or the wild type strains as a way
to compare the signs of the disease (Figure 7). Canker symptoms seemed relatively less
severe in the mutant infection and both strains began to show the disease simultaneously,
by the 5th day. This indicates that PGM could have some role, although not so determinant,
in X. citri subsp. citri pathology.
4
- DISCUSSION
1
3

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Here, we report a characterization of the PGM from X. citri subsp. citri. The protein
structure was solved, functional parameters determined, and its importance on citrus canker
development explored.
As observed in other PGMs [15, 33], the XcPGM active site is located in a cleft
between domains I and IV. In domain I, a conserved phosphoserine residue (SEP119) was
2
+
found phosphorylated in the three structures. Also, all the structures had a bound Mg ion
40]. As previously discussed [41], a striking functional structure named invariant
[
phosphate binding site (IPBS) should be formed in most (if not all) PGMs when in "closed"
conformation, like the XcPGM ligand-bound structures. IPBS involves a set of four
conserved residues placed in a common loop of domain IV plus one residue from domain I
(respectively R421, S423, N424, T425 and Y17 in P. aeruginosa) that get close enough to
hydrogen-bond a phosphate group from the incoming ligand phosphosugar. Additionally, a
sixth residue (K285) is proposed to be recruited on IPBS formation only around 6-
phosphosugar ligand moieties. All these elements (R436, S438, N439, T440 and Y31;
K129 is only found in G1,6P complex) are conserved in XcPGM structure. Also, our
structural data taken together comply with the G1,6P intermediate 180° reorientation
mechanism proposed for phosphohexomutases [18, 33, 40].
Enzyme activity was studied using G1P as substrate, but based on sequence
similarities,
XcPGM
appears
to
be
closer
to
a
bifunctional
phosphomannomutase/phosphoglucomutases (PMM/PGM; EC 5.4.2.8) rather than a strict
PGM [15]. Kinetic parameters vary considerably among PGMs [42] to establish some
narrow rule. As an immediate comparison, the PMM/PGM from Pseudomonas aeruginosa
[
33], the closest sequence homolog (34 % identity, 52 % similarity) with solved structure
1
4

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(
PDB 2FKF), has a K around 27 µM. PGMs K values range from 0.003 mM for
M
M
Toxoplasma gondii [43] to 3.5 mM for Giardia lamblia [16] and XcPGM lies within this
range, being closer to Sphingomonas paucimobilis PGM K of 0.33 mM [17]. Rather than
M
triggering catabolic pathways, relatively high G1P K values are proposed to describe
M
enzyme forms that favor the synthesis of extracellular polysaccharides [44] (like xanthan
for instance), which might be the case.
Although not studied here, it is reasonable to think that the PGM deletion should
affect xanthan synthesis, hence impact on the pathogenicity, due to PGMs key role in the
pathway [23, 45]. Xac∆3579 mutant colony morphology and delayed growth in
pathogenicity-inducing medium XAM-M are consistent with that. However, we found that
PGM did not seem crucial on artificial infection of Citrus aurantifolia leaves. On the
contrary, wild type and mutant infection signs were barely different, although the deletion
had a very evident effect on bacterial growth in vitro. Indeed, Xac∆3579 seemed to cause
somewhat milder canker symptoms but still retained its infectivity. Perhaps this apparent
discrepancy is more related to how much the XAM-M medium or the detached leaves
infection method cannot emulate the natural infection than the relevance of xanA in the
disease. Maybe other infection models, closer to the natural process or that quantify better
the canker infection, could be more suitable to evaluate the influence of PGM on the
disease. Such possibilities might be explored in future works but for now it is reasonable to
speculate that PGM itself is not vital to canker progression.
5
- CONCLUSION
XanA, which encodes a predicted phosphoglucomutase in X. citri subsp. citri is
required to synthesize the pathogenesis-related polysaccharide xanthan. As expected,
1
5

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XcPGM kinetics indicates a role in the synthesis of G1P for precursors of xanthan. High-
resolution structures of XcPGM provide a detailed view of the enzyme’s active site and
indicates the residues involved in catalysis. The deleted mutant clearly suffered impact
from the lack of PGM, but curiously remained able to infect detached citrus leaves.
However, the latter results should be taken with limitations at least due to the artificial
infection methodology. PGM does not seem to be mandatory to the development of citrus
canker symptoms but the impaired xanthan synthesis should affect citrus canker in some
way.
6
- FUNDING
This work was supported by "Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior", CAPES-PNPD Institucional 23038.006942/2011-31 and "Fundação de Amparo
à Pesquisa do Estado de São Paulo", FAPESP Proc. 07/50910-2.
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6

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Figure 1
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0

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A
B
Figure 2
2
1

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Figure 3
2
2

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Figure 4
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3

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Figure 5
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Figure 6
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Figure 7
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Table 1 - Statistics of XcPGM crystals data processing and refinement.
Data Collection
Space Group
Cell dimensions (Å)
a, b, c.
APO
G1P
G16P
P2 2 2
P2 2 2
P2 2 2
1 1 1
1
1
1
1
1
1
4
3.98, 55.18, 174.70
PILATUS 6M
DLS I24
43.90, 54.70, 173.20
43.98, 54.79, 173.50
+
+
Detector
RIGAKU RAXIS IV
RIGAKU RAXIS IV
RIGAKU MicroMax
RIGAKU MicroMax 007
X-ray source
0
07 HF
HF
Wavelength (Å)
Resolution range (Å)
0.96861
1.541
20.00 - 1.85 (1.96 -
1.85)
1.541
5
2.62 - 1.27 (1.30 -
20.00 - 1.85 (1.96 - 1.85)
1.27)
Redundancy
Rmeas (%)*
CC (1/2)
6.3 (5.8)
4.5 (70.7)
3.13 (2.89)
3.71 (2.89)
2.4 (5.0)
2.3 (6.5)
0.999 (0.739)
99.6 (97.8)
1.00 (0.996)
98.4 (92.7)
1.00 (0.997)
97.4 (89.5)
Completeness(%)
Total reflections
Unique reflections
I / (I)
713871 (46694)
112663 (8100)
20.0 (2.8)
135002 (15464)
36040 (5343)
45.99 (17.74)
132372 (14908)
35635 (5146)
45.06 (22.89)
Refinement
parameters
Reflections used for
112557
36037
35633
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refinement
R (%)**
16.48
18.69
3450
0
15.20
17.97
3455
17
14.55
18.51
3450
21
RFree(%)**
No. of protein atoms
No. of ligand atoms
2
B (Å )
1
4.7
13.2
21.80
0.14
14.95
31.13
0.16
Protein
Ligand
---
Coordinate Error (ML
based) (Å)
0.10
o
Phase error ( )
16.82
16.12
16.27
Ramachandran Plot
Favored (%)
97.76
2.24
0.0
97.09
2.68
0.22
2.62
97.09
2.91
0.0
Allowed (%)
Outliers (%)
All-atom Clashscore
RMSD from ideal
geometry
4.10
2.63
r.m.s. bond lengths
0.007
0.004
0.008
(Å)
r.m.s. bond angles (°)
PDB ID
1.118
0.866
0.893
5KL0
5BMN
5BMP
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Highlights:




Xanthomonas citri phosphoglucomutase structure was solved at high resolution;
Structures were also obtained bound with substrates G1P and G1,6P;
Phosphoglucomutase enzyme kinetics was studied using G1P as substrate;
The xanA deleted mutant was obtained and caused milder canker symptoms.
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