Structural and functional characterization of ydji, an aldolase of unknown specificity in escherichia coli K12

Article abstract of DOI:10.1021/acs.biochem.9b00326

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent k_cat of 3.0 s^-1. The product, l-glycero-l-galacto-octuluronate-1-phosphate, has a k_cat/K_m value of 2.1 × 10³ M^-1 s^-1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l-glycero-l-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 ? (Protein Data Bank entry 6OFU), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l-glycero-l-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l-glycero-l-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Full text of DOI:10.1021/acs.biochem.9b00326

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Article
Structural and Functional Characterization of YdjI, an
Aldolase of Unknown Specificity in Escherichia coli K12
Jamison P Huddleston, James B. Thoden, Brandon J Dopkins, Tamari
Narindoshvili, Blair J Fose, Hazel M. Holden, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00326 • Publication Date (Web): 19 Jul 2019
Downloaded from pubs.acs.org on July 24, 2019
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Structural and Functional Characterization of YdjI, an
Aldolase of Unknown Specificity in Escherichia coli K12
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‡
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Jamison P. Huddleston , James B. Thoden , Brandon J. Dopkins , Tamari
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Narindoshvili , Blair J. Fose , Hazel M. Holden , and Frank, M. Raushel *
‡Department of Chemistry, Texas A&M University, College Station, Texas, 77843, United
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δDepartment of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin,
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ABSTRACT
The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and
other closely related species in the human microbiome. Based on the annotations of the
enzymes located in this cluster, it is expected that together they catalyze the catabolism of
an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj
gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function.
Here we describe a structural and functional characterization of this enzyme. YdjI
catalyzes the hydrogen/deuterium exchange of the proS hydrogen at C3 of
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dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol
condensation with a variety of aldo-sugars. YdjI shows strong preference toward higher-
order (seven-, eight- and nine-carbon) monosaccharides with specific hydroxyl
stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best
-1
substrate is L-arabinuronic acid with an apparent k = 3.0 s . The product, L-glycero-L-
cat
3
-1 -1
galacto-octuluronate-1-phosphate, has a value of k /K of 2.1 × 10 M s in the retro-
cat
m
aldol reaction with YdjI. This is the first recorded synthesis of L-glycero-L-galacto-
octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI,
determined to a nominal resolution of 1.75 Å (PDB code: 6OFU), reveals unusual positions
for two arginine residues located near the active site. Computational docking was utilized
to distinguish preferable binding orientations for L-glycero-L-galacto-octuluronate-1-
phosphate. These results indicate a possible alternative binding orientation for L-glycero-L-
galacto-octuluronate-1-phosphate than observed in other class II aldolases, which utilize
shorter carbohydrate molecules.
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INTRODUCTION
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There are an estimated 10 microbial cells living within every human whose
combined number of genes is predicted to outnumber human genes by two-orders of
magnitude (1). A growing body of evidence suggests that individual human health is
dependent upon a symbiotic relationship amongst the variety of bacterial species
comprising the human microbiome (2, 3). Indeed, a number of disease states, such as
colorectal cancer, diabetes, obesity, and cardiovascular problems, have been linked to a
dysbiosis, or an imbalance, in the human gut microbiota (4). One example is the
pathogenesis of inflammatory bowel disorders, specifically Crohn’s disease and ulcerative
colitis, with colonization of adherent-invasive Escherichia coli (AIEC) (5). Strikingly,
prevention of Crohn’s disease regression is positively influenced by colonization of
Faecalibacterium prausnitzii (6). However, the exact mechanisms behind the prevention
and progression of chronic colitis, and the source of the dysbiosis between these species in
an individual’s gut are unknown.
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One possible explanation is that metabolic differences amongst gut microbiota allow
for certain species to gain a selective advantage over others. Humans do not metabolize or
absorb most of the carbohydrates consumed (7). However, bacterial species in the human
gut have evolved the ability to metabolize many of these carbohydrates (8). Indeed, this is
one mechanism by which both commensal and pathogenic species enhance their
colonization (9-11). For commensal species, this is a protection mechanism from harmful
pathogenic species known as colonization resistance (10, 12). To date, however, the
metabolic utilization of alternative carbohydrate sources of many of these bacterial species
remains poorly characterized.
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Many carbohydrate catabolic pathways utilize an aldolase in the final step (13, 14).
Specifically, aldolases, which are essential and ubiquitous, catalyze the stereospecific
formation or breaking of carbon-carbon bonds (13, 14). The most well-known example is
D-fructose-1,6-bisphosphate aldolase (FbaA), which catalyzes the reversible cleavage of D-
fructose-1,6-bisphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-
phosphate (Scheme 1) (15). Other well-studied aldolases include D-tagatose-1,6-
bisphosphate aldolase (KbaY and GatY), L-rhamnulose-1-phosphate aldolase (RhaD), L-
fuculose-1-phosphate aldolase (FucA), and 2-deoxy-5-keto-D-gluconate-6-phosphate (IolJ),
which is essential for myo-inositol degradation in Bacillus subtilis (16, 17).
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Scheme 1: Reaction catalyzed by D-fructose-1,6-bisphosphate aldolase.
Common aldolases are divided into two classes based on the mechanism by which
the reactions are catalyzed (15). Class I aldolases utilize an active site lysine residue to
form a Schiff-base intermediate. Class II enzymes employ an active site transition metal,
usually zinc, to stabilize an enolate intermediate. These two classes of enzymes evolved
separately and are mechanistically unrelated. Class II aldolases are subdivided into two
clusters of orthologous groups (COGs) (18). COG0191 contains, for example, FbaA, KbaY,
GatY, and IolJ, whereas RhuD and FucA are found in COG0235. E. coli K12 has four
enzymes currently annotated as members of COG0191, and three of these enzymes have
been well-characterized. One is fructose-1,6-bisphosphate aldolase used in glycolysis
(FbaY; Uniprot id: P0AB71); the other two are tagatose-1,6-bisphosphate aldolases (GatY;
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Uniprot id: P0C8J6 and KbaY; Uniprot id: P0AB74). The fourth example is YdjI (Uniprot id:
P77704), an enzyme of unknown function.
YdjI is localized in a cluster containing genes encoding five enzymes, two
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transporters, and a regulator (Figure 1). Based on the putative annotations of these
enzymes, this cluster is likely to be responsible for the catabolism of a carbohydrate of
unknown structure. In this gene cluster, YdjL and YdjJ are zinc-dependent alcohol
dehydrogenases. YdjH is annotated to be in the ribokinase family (19). YdjG is an aldo-
keto reductase family member, which was previously shown to catalyze the reduction of
methyl glyoxal to hydroxyacetone (20). YdjK and YdjE are members of the major facilitator
superfamily of transporters (19). YdjF is a helix-turn-helix, Deo-R type transcriptional
regulator (19, 21). This gene cluster is highly conserved amongst E. coli strains, appearing
in approximately 80% of sequenced E. coli genomes including: E. coli K12, the probiotic
strain E. coli Nissle 1917, the adherent-invasive E. coli LF82, and the enterohemorrhagic E.
coli O157:H7 (21, 22). It is also found in species that are closely related to E. coli including
the Gram-negative Shigella dysenteriae and the Gram-positive Clostridium bolteae.
Figure 1. Organization of the ydj gene cluster in E. coli. The cluster includes genes
encoding five enzymes: YdjL, YdjJ, YdjI, YdjH, and YdjG (shown in blue), two transporters:
YdjK and YdkE (shown in green), and a transcriptional regulator, YdjF (shown in red).
The ydj gene cluster was discovered as part of our larger initiative to identify
carbohydrate catabolic pathways with unknown functions. Enzymes of potential interest
were identified by first searching species of the human gut microbiome for proteins
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commonly found in carbohydrate catabolic pathways as predicted by their COG
assignments. A computer script was written to retrieve the genes of interest from selected
species and then the sequences were compared to a self-compiled database of known
carbohydrate catabolic enzymes based on annotation scores from the Uniprot database
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(
23). This computational approach of identifying uncharacterized carbohydrate catabolic
pathways is fast, less dependent on high-quality genome annotation and offers the
advantage of considering genomic context of an enzyme without adding additional
complexity. As an initial validation for this methodology, the E. coli K12 genome was
interrogated and four potential unknown carbohydrate catabolic pathways were identified
with the ydj gene cluster being one. The initial goal was to quickly characterize the five
enzymes of the gene cluster, but complete characterization of this metabolic pathway has
remained elusive. Here we describe the functional characterization of the substrates and
three-dimensional structure of YdjI, the remaining uncharacterized class II aldolase from E.
coli K12.
EXPERIMENTAL METHODS
Materials and Equipment. All materials used in this study were obtained from
Sigma-Aldrich, Carbosynth, or GE Healthcare Bio-Sciences, unless stated otherwise. E. coli
strains XL1 Blue and BL21-Gold (DE3) were obtained from New England Biolabs. NMR
spectra were collected on a Bruker Avance III 400 MHz system equipped with a broadband
probe and sample changer, or a Bruker Avance 500 MHz system equipped with a triple
resonance cyroprobe. Mass spectrometry samples were collected using an MDS-Sciex
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000 Qtrap system or a Thermo Scientific Q Exactive Focus system run in negative ion
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mode. UV spectra were obtained with a SpectraMax340 UV-visible plate reader and 96-
well NucC plates.
Sequence Similarity Networks. Sequence similarity networks (SSN) were
generated by submitting the InterPro number for the protein family of interest or a list of
the Uniprot IDs assigned to an individual Cluster of Orthologous Genes (COG) to the EFI-
EST webtool (24). The initial SSN was generated with an alignment score cutoff set such
that each connection (edge) represents approximately 30% sequence identity. Sequences
that share high sequence identity (>90%) were grouped into a single node. All network
layouts were created and visualized using Cytoscape 3.4 (25). Within Cytoscape, more
stringent SSNs were created by increasing the alignment score cutoff in small increments
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(
usually by 5-10). This process was continued until the clusters were estimated to be
isofunctional, where each cluster represents enzymes that catalyze the same reaction.
Isofunctionality was determined by mapping the known functions onto the SSNs and
following their movements as the alignment cutoff is increased until each cluster contains a
single known catalytic activity.
Cloning of YdjI from E. coli K12. The gene for YdjI (Uniprot id: P77704) was
amplified from the genomic DNA of E. coli K12 using the primer pair:
5
ʹ-ACGCATATGCTCGCAGATATCAGATATTGGGAAAACGATG-3ʹ
ʹ-ATTAAGCTTTTCCGCTTTGCCATCAGAACCGAACA-3ʹ
5
Restriction sites for NdeI and HindIII (underlined) were introduced into the forward and
reserve primers, respectively. These sites allow for the addition of a C-terminal 6x-His-tag
in the pET-30a+ expression vector.
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PCR amplification was carried out with Phusion DNA polymerase following the
manufacture specifications. Amplified PCR products were purified using the QIAquick PCR
purification kit from Qiagen. Subsequently, restriction enzymes (New England Biolabs)
and CutSmart buffer were added and allowed to incubate for 2 h at 37 °C. Digested genes
were purified using agarose (1%) gel electrophoresis and the QIAquick gel extraction kit
from Qiagen. The pET-30a vector (Novagen) was subjected to the same restriction enzyme
digestion and gel purification. Additionally, the 5ʹ-end phosphate was removed by
incubating with recombinant shrimp alkaline phosphatase (rSAP) for 2 h after restriction
digest. The genes were cloned into the digested vector using DNA ligase (New England
BioLabs) following the supplied protocol. After ligation, high salt buffer was removed by
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the PCR purification kit and eluted with 20 µL of deionized H O. The resulting plasmid
2
(
2 µL) was used to transform E. coli XL1-blue cells (40 µL) via electroporation, and the cells
allowed to recover in 2 mL of lysogeny broth (LB) medium at 37 °C. After 2 h, the cells
were harvested by centrifugation, re-suspended in 100 µL of LB medium, and plated on
kanamycin selective LB medium. Individual colonies were selected and grown in 5 mL of
liquid LB medium overnight at 37 °C and recombinant plasmids were harvested using
GenElute Plasmid Miniprep Kit from Sigma-Aldrich. Plasmids were sequenced to identify
the correct cloned genes in the appropriate vector.
Expression and Purification of YdjI. Recombinant plasmids containing the ydjI
gene were used to transform E. coli BL21 (DE3) cells via a heat-shock method and the cells
were plated on LB medium containing 30 mg/L of kanamycin (26). A single colony was
selected to inoculate a 20-mL starter culture and grown overnight at 37 °C. This culture
was used to inoculate 2 L of LB medium (1.0 L of medium in 2 x 2.8-L Fernbach flasks) and
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grown at 37 °C until the OD600 = 0.6 was obtained. Zinc acetate (1.0 mM) was added to the
growth medium when the OD600 = ~0.1-0.2. Protein expression was induced by the
addition of 1.0 mM isopropyl β-thiogalactoside (IPTG) at 37 °C for 4-5 h. Cells were
harvested by centrifugation at 8000 g for 10 min. Typically, cells pellets (~3 g) were frozen
and stored at -80 °C until purification.
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Frozen cells were resuspended in 50 mL of 50 mM HEPES/K , pH 8.5 with 250 mM
KCl and 10 mM imidazole and then lysed with at least seven rounds of sonication at 4 °C.
DNA and RNA were degraded by the addition of 1.0 mg of bovine DNase and RNase
followed by stirring on ice for 10 min. Cell debris was removed by centrifugation at
1
2,000 g for 40 min followed by syringe filtering with a 0.2 µm filter. The resulting
supernatant solution was loaded onto a 5-mL HisTrap HP (GE Healthcare) nickel affinity
column that had been pre-equilibrated with resuspension buffer using a BioRad FPLC
+
system. Protein was eluted from the column using 50 mM HEPES/K , pH 8.5, with 250 mM
KCL and 500 mM imidazole over a gradient of 30 column volumes. Fractions of 4.0 mL
were collected and tested for purity by SDS-polyacrylamide gel electrophoresis. Typically,
protein eluted between 8-15 column volumes. Fractions containing the purest samples
+
were pooled and buffer exchanged by dialysis into 50 mM HEPES/K , pH 8.5 with 250 mM
KCl overnight at 4 °C. Homogenous protein was concentrated using Vivaspin 20 centrifugal
concentrators (GE Healthcare) with the final concentration of enzyme determined by the
-1
-1
absorbance at 280 nm. The extinction coefficient (E280 = 30,440 M cm ) was estimated
based on the protein sequence including the C-terminal His-tag and linker (27). Typically,
protein was divided into 100 µL aliquots, flash frozen, and stored at -80 °C. Yields were
typically 75-100 mg of YdjI from 2.0 L of cell culture. The metal content was determined
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using ICP-MS. For this, purified protein was digested with concentrated nitric acid by
refluxing for 2 h and then diluted with metal-free water to give a final nitric acid
concentration of 1% (v/v).
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Synthesis of Noncommercial Carbohydrates. The methods for the synthesis of
compounds not commercially available are found in the Supplementary Information.
These compounds include: penturonic acids (D-arabinuronate, L-arabinuronate, D-
xyluluronate, L-lyxuronate, and D-riburonate), teturonic acids (L-threouronate, D-
threouronate, and D-erythruronate), DHAP, hydroxyacetone phosphate, tartronate
semialdehyde, glycoaldehyde, L-threose-4-P, D- and L-arabinose-5-P, and D-xylose-5-P.
YdjI-Catalyzed Solvent-Deuterium Exchange. YdjI was placed in 25 mM
imidazole-D O buffer using Vivaspin 500 concentrators with 10 kDa cut-off filters.
2
Potential substrates for YdjI (10 mM) were mixed with enzyme (10 µM) in 20 mM
imidazole-D O buffer, pH 7.4. The compounds assayed for catalytic activity included
2
pyruvate, dihydroxyacetone, dihydroxyacetone phosphate, hydroxypyruvate,
hydroxyacetone, hydroxyacetone phosphate, methylglyoxal, and acetone. Exchange of the
1
α-protons with deuterium from solvent was monitored by H NMR spectroscopy for up to
48 h. For measurement of the rate of solvent exchange with DHAP catalyzed by YdjI,
1
1
0 mM DHAP was mixed with 2 µM YdjI and the H NMR spectrum was collected every 90 s
for 60 min. The extent of substrate exchange with solvent was calculated from the peak
area with respect to an internal standard of acetate. The change in the concentration of
DHAP over time was plotted and fit to eq. 1, where λ is the observed first-order rate
constant for solvent exchange with the substrate. The turnover rate constant (k ) was
cat
determined using eq. 2, assuming a saturating substrate concentration.
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-λt
A /A = e + C
(1)
(2)
t
o
kcat = λ [A]/E
t
Stereochemistry of H/D Exchange in DHAP Catalyzed by YdjI. DHAP (10 mM)
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was added to YdjI (2 µM) into 25 mM imidazole-D O buffer, pH 7.4. Sodium acetate (5.0
2
mM) was added as a calibration standard. The reaction was allowed to proceed for 2 h,
until the DHAP was completely exchanged with deuterium from solvent. Commercial
rabbit muscle aldolase (Sigma-Aldrich) was diluted into 25 mM imidazole buffer to a
1
concentration of 1 U/µL and 1.0 U was added to the reaction above and monitored by H
NMR spectroscopy for an additional 4 h.
pH Dependence of H/D Exchange of DHAP Catalyzed by YdjI. The exchange
reactions were conducted starting from 500 mM imidazole-D O buffer stocks at pH 6.0, 6.5,
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.0, 7.5, and 8.0 by adding DCl until the desired pH was obtained. These concentrated
buffers were diluted to 25 mM imidazole-D O working stocks. DHAP (5.0 mM) was added
2
to YdjI (2.0 µM) into 25 mM imidazole-D O buffer, pH 6.0-8.0. The reactions were followed
2
1
by H NMR spectroscopy by collecting a spectrum every 120 s for 30 min. The final pH of
reaction was measured after the reaction was complete. The change in the concentration
of DHAP over time was plotted and fit to eq. 1 to yield an apparent rate of turnover as
described above.
YdjI Assays for Catalytic Activity. YdjI was screened for catalytic activity using
two methods. The aldol condensation reaction was monitored by mixing 10 mM DHAP,
1
0 µM YdjI, and 15 mM potential aldehyde substrates in 25 mM imidazole-D O buffer,
2
+
31
pH 7.4 or 50 mM HEPES/K , pH 7.4, and monitoring the reaction as a function of time by P
1
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or H NMR spectroscopy at 32 C. For experiments containing DHAP and phosphorylated
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sugars, 10 mM of each substrate was used to minimize the complexity of the final NMR
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spectra. Reactions were followed by P NMR spectroscopy for 24 - 72 h. Kinetic
experiments were conducted under the same reaction conditions as the original assays
with a variable concentration of YdjI (2-10 µM) depending on the observed rate of
turnover. Spectra were collected every three minutes for 1.0 h with the product
resonances quantified by integration with respect to an internal standard (phosphate or
methyl phosphonate). Concentration of product was plotted as a function of time and fit to
a linear equation or a single exponential.
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The retro aldol reaction was monitored by UV spectroscopy using a coupled assay
containing 5.0 µM YdjI, 1-10 mM substrate, glycerol-3-phosphate dehydrogenase (G3PD,
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.1 U), and 300 µM NADH at 30 C. If DHAP was not a product of this transformation (e.g.
fructose-6-phosphate), triose phosphate isomerase (1 U) was included to convert D-
glyceraldehyde-3-P to DHAP. The values of k and k /K were determined by fitting the
cat
cat
m
initial velocity data to eq. 3 using GraFit 5, where v is the initial velocity of the reaction, E is
the enzyme concentration, k is the turnover number, and K is the Michaelis constant.
cat
m
v/E = k A/ (K + A)
(3)
t
cat
m
Stereochemistry of YdjI Catalyzed Reaction. YdjI was used to enzymatically
synthesize fructose-1,6-bisphosphate (FBP). DHAP was generated in situ using 50 mM
dihydroxyacetone, 1.0 mM ATP, 2.0 mM MgCl and 1 U of glycerol kinase. The ATP was
2
recycled using 60 mM PEP and 5 U of pyruvate kinase. DHAP was converted to G3PD using
triosephosphate isomerase (100 U). To generate FBP, 20 µM YdjI was added to a final
+
reaction volume of 1.0 mL in 50 mM HEPES/K buffer, pH 8.0. The reaction was monitored
3
1
for 24 h using P NMR. For purification of FBP, enzymes were removed using a PALL-
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Omega 10K centrifugal filtration column. The eluent flow-through was loaded onto a DEAE
Sephadex A25 column (1.5 x 10 cm) that had been pre-equilibrated with 500 mM NH HCO ,
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pH 8.5, and washed with water until the pH was neutral. FBP was eluted from the column
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using a 100-mL gradient of 0-500 mM NH HCO . Fractions were monitored using the
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phenol-sulfuric acid test for carbohydrates with the most concentrated fractions pooled
and concentrated under reduced pressure (28, 29). The NMR spectrum of the purified FBP
was compared to that for commercially obtained FBP and comparison of catalytic activities
with commercially obtained FBP aldolase using the coupled assay described above for the
retro-aldol reaction.
Purification of YdjI Catalyzed Reaction Products. Selected products of the YdjI
catalyzed reactions were purified so they could be further characterized or assayed. After
the reactions were complete, YdjI was removed using a PALL-Omega 10K centrifugal
filtration column. The flow-through was purified using DEAE anion-exchange
chromatography following the procedure outlined above. Depending on the number of
negative charges, most compounds eluted between 100-250 mM NH HCO . Fractions were
4
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tested for the presence of carbohydrates using the phenol-sulfuric acid test. Those that
+
tested positive were pooled and acidified using Dowex H resin to a pH of 3-4 to remove the
ammonium bicarbonate buffer. The Dowex resin was removed by filtration, and the flow-
through was neutralized to a pH of 6.0 using NaHCO and concentrated to dryness under
3
reduced pressure.
Crystallization and Structural Analysis. For crystallization purposes, the gene
encoding YdjI was re-cloned and ligated into pET-31(b+) to generate a construct with a C-
terminal polyhistidine tag (LEHHHHHH). The pET-31-ydjI plasmid was utilized to
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transform Rosetta2(DE3) E. coli cells (Novagen). The cultures were grown in lysogeny
broth supplemented with ampicillin (100 mg/L) and chloramphenicol (50 mg/L) at 37 oC
with shaking until an optical density of 0.5 was reached at 600 nm. At this time zinc acetate
was added (0.1 mM), and the growth allowed to continue until the optical density reached
approximately 0.8. The flasks were cooled in an ice bath, and protein expression was
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initiated with the addition of 1.0 mM IPTG. The cells were then allowed to grow at 22 C for
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8 h. The cells were harvested by centrifugation and disrupted by sonication on ice. The
lysate was cleared by centrifugation, and YdjI was purified with PrometheusTM Ni-NTA
agarose (Prometheus Protein Biology Products) according to the manufacturer’s
instructions. The protein was dialyzed against 10 mM Tris-HCl, pH 8.0, and 200 mM NaCl,
-1
-1
and concentrated to 25 mg/mL based on an extinction coefficient of 0.99 (mg/mL) cm at
2
80 nm.
Crystallization conditions for YdjI were surveyed at both room temperature and 4 oC
by the hanging drop method of vapor diffusion using a laboratory-based sparse matrix
screen. X-ray diffraction quality crystals of the enzyme were subsequently grown from
precipitant solutions containing 10 to 12% poly(ethylene glycol) 5000, methyl ether and
o
100 mM MES (pH 6.0) at 21 C in the presence of 5.0 mM DHAP. The crystals belonged to
the space group P2 with unit cell dimensions of a = 80.8 Å, b = 85.7 Å, and c = 84.3 Å and 
1
o
=
109.9 . The asymmetric unit contained four subunits.
For X-ray data collection, the crystals were transferred to a cryoprotectant solution
containing 22% poly(ethylene glycol) 5000 methyl ether, 300 mM NaCl, 5.0 mM DHAP,
6% ethylene glycol, and 100 mM MES (pH 6.0). X-ray data were collected at the Advanced
Photon Source, Structural Biology Center Beamline 19-BM. The X-ray data were processed
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with HKL3000 (30). The structure was solved via molecular replacement with PHASER
using as a starting model the structure of tagatose-1,6-bisphosphate aldolase (PDB code:
1GVF) (31, 32). Iterative cycles of model building with COOT and refinement with REFMAC
reduced the Rwork and Rfree to 14.1% and 18.2%, respectively, from 50 to 1.75-Å resolution
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(33-35). Relevant X-ray data collection and model refinement statistics are presented in
Table 1.
Computation Docking of Ligands to YdjI. Computational docking was conducted
using the AutoDock Vina plugin (V2.1.1) for Pymol (V1.8 or higher) or using the command-
line scripts from AutoDock Tools Package provided by The Scripps Research Institute (37-
40). The receptor was chosen to be chains A and B in the X-ray coordinate file. Chains A
and B served as the active site and the secondary subunit, respectively. The search space
was confined to a 24 Å × 20 Å × 27 Å box, centered near residue Phe52 of chain A, and sized
such that two arginine residues, Arg84 and Arg254ʹ, from the adjacent chain B subunit,
could be included as flexible residues. The docking output was set to provide 10 poses. A
modified version of the flexible side chain method for covalent docking technique was used
after the initial AutoDock Vina plugin failed to produce realistic results with DHAP and L-
glycero-L-galacto-octuluronate-1-P (41). For the docking optimization of L-glycero-L-
galacto-octuluronate-1-P, DHAP was manually positioned into the YdjI structure using an
overlay of bound DHAP from a homologous structure (PDB: 5VJD) (13). The DHAP moiety
was then included as part of the receptor, held fixed, and the hydrate of L-arabinuronate
was docked using AutoDock Vina. The top results were manually positioned near the
DHAP moiety and a bond between C3 of DHAP and C1 of L-arabinuronate was created in
silico, to yield L-glycero- L-galacto-octuluronate-1-P. The resulting 8-carbon
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monosaccharide was designated to be part of a new receptor file and allowed to act as a
flexible side chain with all bonds between C1, C2, C3, and C4 held fixed and inactive during
the docking routine. The remaining bonds of L-glycero-L-galacto-octuluronate-1-P were
allowed to be optimized during the docking calculations. For this experiment, a water
molecule served as the ligand to be bound.
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Table 1. X-ray Data Collection and Refinement Statistics.
resolution limits
50 – 1.75
b
(
1.78 – 1.75)
space group
P2
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2
2
2
2
2
2
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3
3
3
3
3
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3
3
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5
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6
0
1
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unit cell dimensions
a (Å)
b (Å)
c (Å)
80.8
85.7
84.3
o
 ( )
109.9
number of independent
reflections
104686 (7205)
96.5 (90.6)
completeness (%)
redundancy
3.6 (2.6)
33.2 (4.4)
7.1 (21.3)
avg I/avg (I)
a
Rsym (%)
c
R-factor (overall)%/
14.3/104686
no. reflections
R-factor (working)%/
no. reflections
R-factor (free)%/
14.1/99486
18.2/5200
no. reflections
number of protein atoms
number of heteroatoms
average B values
8142
1006
2
protein atoms (Å )
24.1
43.9
34.5
2
metals (Å )
2
solvent (Å )
weighted root-mean-square
deviations from ideality
bond lengths (Å)
0.011
1.4
0.009
o
bond angles ( )
o
general planes ( )
Ramachandran regions (%)
most favored
d
99.0
1.0
0.0
additionally allowed
generously allowed
PDB entry
6OFU
a
Rsym = (∑|I - I|/ ∑ I) x 100.
Statistics for the highest resolution bin.
b
c
R-factor = (|F - F | / |F |) x 100 where F is the observed structure-factor amplitude and F is the
o
c
o
o
c
calculated structure-factor amplitude.
Distribution of the Ramachandran angles according to PROCHECK (36).
d
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Bioinformatic Analysis of YdjI. YdjI is assigned to COG0191, which is comprised of
2
280 unique protein sequences as of 01/03/2019. The SSN for COG0191 with the
functionally characterized enzymes and proteins of interest, mapped at two different
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alignment score cutoffs, 55 and 100, is presented in Figure S1. At an arbitrary cutoff of 55
(
~34% sequence identity), YdjI is found in the major cluster containing tagatose-1,6-
bisphosphate aldolase from E. coli (GatY and KbaY) and 2-deoxy-5-keto-D-gluconate-6-
phosphate aldolase (IolJ) from B. subtilis. At a cutoff of 100 (~60% sequence identity), the
SSN is essentially isofunctional, indicating that each cluster likely represents a single
substrate profile. Here, YdjI is found in a small cluster containing six nodes representing a
total of 19 enzymes. In this small cluster of enzymes, all nodes share at least 70% sequence
identity and are found in the same genomic context and gene cluster as YdjI from different
bacterial species including E. coli, S. dysenteriae, S. enterica, and C. bolteae.
Stereospecific Hydrogen Exchange. The gene encoding YdjI from E. coli K12 was
cloned, expressed, and the enzyme purified to homogeneity. ICP-MS analysis showed that
2+
YdjI contained 0.90 ± 0.03 equivalents of Zn per monomer. Eight ketones were screened
as possible substrates for the aldol condensation reaction by monitoring hydrogen
1
exchange with deuterium from solvent by H NMR spectroscopy. Pyruvate,
hydroxypyruvate, methyl glyoxal, acetone, hydroxyacetone, dihydroxyacetone,
hydroxyacetone phosphate, and dihydroxyacetone phosphate (DHAP) were all tested as
potential substrates for YdjI (Figure S2). The hydrogen exchange reactions for each
compound were monitored for up to 72 h, but measurable catalytic activity was only
observed with DHAP.
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Figure 2. Solvent-deuterium exchange within DHAP. (a) H NMR spectrum of DHAP in 25
1
mM imidazole-D O buffer, pH 7.4. (b) H NMR spectrum of DHAP (10 mM) after a 21 min
2
1
incubation with YdjI (2.0 µM). (c) H NMR spectrum of DHAP (10 mM) after a 90 min
1
incubation with YdjI (2 µM). (d) H NMR spectrum of DHAP (10 mM) after a 4 h incubation
with YdjI (2.0 µM) and FBP aldolase (1.0 U). The reaction is illustrated in Scheme 2.
In solution, DHAP exists in two inter-exchangeable forms, where 37% of the keto
group is hydrated (Figure 2a) (42). Hydrogen/deuterium exchange at C3 of DHAP was
observed by following the disappearance of the hydrogen resonance for one of the
prochiral hydrogens at 3.55 ppm and the appearance of a broader resonance at 3.53 ppm.
Alternatively, exchange of a hydrogen at C3 of the keto-form of DHAP was monitored at
.54 ppm. The apparent rate constant for the hydrogen exchange reaction was 8.4 ± 0.3 s-1
4
(
Figures 2b and 2c). To determine which of the two prochiral hydrogens at C3 of DHAP is
exchanged by YdjI, rabbit muscle aldolase was added to the reaction mixture. After an
incubation period of 4 h, no additional hydrogen exchange with solvent was observed
(
Figure 2d). For comparison, 1.0 U of rabbit muscle aldolase was shown to catalyze the
exchange of one of the C3 hydrogens of DHAP (10 mM) in 60 min (data not shown).
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1
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2
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2
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5
6
Therefore, YdjI catalyzes the removal of the same prochiral hydrogen from DHAP as rabbit
FBP aldolase and thus the proS hydrogen from C3 is abstracted (Scheme 2) (14, 43). The
effect of changes in pH on the rate of deuterium-exchange was also examined. YdjI was
shown to be most active at pH 7.1 with a 1.6-fold loss in activity at pH 6.1 and a 3.6-fold
loss in activity at pH 8.0 (Figure S9).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2: Solvent-deuterium exchange reaction within DHAP catalyzed by YdjI.
Product Stereochemistry at C4. The stereochemistry at C4 after condensation
with an aldose sugar remained to be defined. The reaction can occur similar to that
catalyzed by FBP aldolase, resulting in a product with R-stereochemistry at C4, or
alternatively, with the opposite stereochemistry in a reaction similar to that catalyzed by
tagatose-1,6-bisphosphate (TBP) aldolase (43). YdjI was mixed with DHAP and D-
glyceraldehyde-3-P (D-G3P) to generate either FBP or TBP. DHAP was generated in situ
using glycerol kinase, ATP and dihydroxyacetone. D-G3P was generated from DHAP by the
addition of triose phosphate isomerase (TIM). Upon addition of YdjI, FBP is the primary
3
1
product, although small amounts of uncharacterized side products are visible in the P
NMR spectrum (Figure 3a). The same reaction with FBP aldolase produces the α- and β-
anomeric forms of FBP (Figure 3b). To demonstrate that the reaction product is FBP, it
was purified by anion exchange chromatography (Figure 3c) and compared with FBP
obtained commercially (Figure 3d). The YdjI-generated FBP was also demonstrated to be
a substrate for FBP aldolase when the reaction was monitored by a coupled spectroscopic
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assay. Therefore, the YdiI catalyzed condensation of DHAP and D-G3P produces FBP with
formation of an S-stereocenter at C3 and an R-stereocenter at C4.
1
1
1
1
1
1
1
1
1
1
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. 31P NMR spectra of YdjI and FPB aldolase reaction products. (a) Reaction of YdjI
with DHAP and TIM to generate D-fructose-1,6 bisphosphate (FBP). (b) Reaction of FBP
aldolase with DHAP and TIM to generate FBP. (c) FPB purified from the reaction mixture
3
1
depicted in Figure 3a. (d) P NMR spectrum of FBP obtained commercially. Peak
assignments for FBP obtained from previously published reports (44, 45).
Screening for Aldose Sugar Substrates. After determining that DHAP is the ketose
component for the YdjI-catalyzed reaction, potential aldose fragments for the condensation
1
31
reaction were screened by H and P NMR spectroscopy. In total, 49 compounds were
tested as potential substrates for YdjI in the presence of DHAP (Figure S3). It was initially
determined that YdjI will accept methyl glyoxal (13) as a substrate with formation of 6-
deoxy-5-keto-fructose-1-P (DKFP, 40). Methyl glyoxal is a degradation product of DHAP
and a common contaminate in commercially available DHAP (46, 47). Unlike the high
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specificity for the keto-substrate, significant promiscuity was observed within the
aldehyde-containing substrates. YdjI catalyzed the condensation of 33 different aldose
substrates with DHAP (Figure 4). Activity was determined by observation of NMR
resonances that could be readily distinguished from those of either DHAP or DKFP (40).
The compounds that showed the most robust activity had the turnover numbers quantified
0
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by following the reaction over time using P NMR spectroscopy (Table 2). The isolated
products were subjected to mass spectrometry and were confirmed to have the expected
molecular weight (Figure 5).
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0
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8
9
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Figure 4: Structures of aldoses found to be substrates for YdjI in the aldose condensation
reaction with DHAP.
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Table 2. Turnover numbers for substrates with YdjI and DHAPa
Found
apparent
Expected
Product
MW
(M)
Substrate
MW
M-H)
-1 b
k
cat (s )
-
(
3
5
6
34
35
0.40 ± 0.05
2.2 ± 0.2
243.99
244.03
244.03
274.00
242.01
290.03
290.03
304.01
370.00
334.02
334.02
334.02
400.01
400.01
364.03
364.03
242.99
243.03
243.03
273.00
241.01
289.03
289.03
303.01
369.00
333.02
333.02
333.02
399.01
399.01
363.03
363.03
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
4.8 ± 0.6
9
/10
37/38
40
0.50 ± 0.02
0.64 ± 0.03
0.71 ± 0.06
0.48 ± 0.04
0.68 ± 0.04
0.66 ± 0.04
3.0 ± 0.3
1
3
4
5
7
1
1
1
41
42
43
20
25
26
28
29
30
32
33
44
45
46
2.3 ± 0.2
47
>0.7c
48
0.50 ± 0.04
1.60 ± 0.09
0.050 ± 0.004
1.8 ± 0.1
49
50
51
aReactions were monitored by 31P-NMR spectroscopy and
quantified by integration with an internal phosphate standard.
b
Errors were calculated from the standard deviation of the
c
fitting results as described. Overlap between substrate and
product peaks prevented accurate integrations. The value
reported is a lower limit based on the amount of product
formed in 30 min and corrected for enzyme concentration.
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0
Figure 5. Structures of products formed from the condensation of DHAP with selected
aldehyde substrates by YdjI.
To measure the specificity constant (k /K ) for each substrate, selected
cat
m
compounds were synthesized on a larger scale (~50 mg) by mixing YdjI and DHAP with an
excess of the aldose substrate, and the products purified by anion-exchange
chromatography. These compounds were used as substrates for YdjI in the retro-aldol
reaction. The formation of DHAP was monitored in a coupled enzyme assay with glycerol-
3
-dehydrogenase (Table 3).
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Table 3. Steady-state kinetic parameters of selected
substrates with YdjI
a
-
1
-1 -1
Substrate
k
cat (s )
m
kcat /K (M s )
D-fructose-1-Pb
0.042 ± 0.003
0.060 ± 0.001
<0.010
7.8 ± 1.0
0
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0
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1
2
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7
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0
3
9b
0c
30.5 ± 0.7
4
-
4
1c
<0.001
-
4
2b,c
<0.001
-
4
4
5
6
7
0
1
0.011 ± 0.001
0.860 ± 0.002
0.021 ± 0.012
0.260 ± 0.084
0.038 ± 0.001
0.089 ± 0.002
26.6 ± 3.5
4
(2.1 ± 0.1) × 103
14.7 ± 1.9
4
4
5
5
(4.2 ± 1.6) × 102
(2.0 ± 0.2) × 102
(2.0 ± 0.2) × 102
a
Errors were calculated from the standard deviation of
b
the fitting results. Commerically purchased
compounds. Estimated based on lower limit of
c
detection.
Structural Characterization of Selected Substrates. To further demonstrate that
the proposed stereochemistry at C4 is consistent with the broad substrate profile of YdjI,
two additional experiments were conducted. In the first experiment, the retro-aldol
activities of the well-characterized FBP aldolase with some of the products generated
enzymatically by YdjI were quantified. The high-degree of promiscuity for aldolase
enzymes in the aldol condensation reaction is well-documented (15, 16, 43). However, the
retro-aldol reaction has been shown to be quite selective for a specific stereochemical
arrangement at C3 and C4 (16). For example, FBP aldolase has a 1400-fold higher
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specificity for cleavage of FBP (39) relative to TBP (43). FBP was shown to be a relatively
-1 -1
poor substrate for YdjI with a k /K = 30 M s (Table 3).
cat
m
Six compounds synthesized by YdjI (D-xyluluronate-1-P (34), D-glycero-L-galacto-
octuluronate-1-P (46), L-glycero-L-galacto-octuluronate-1-P (45), L-glycero-D-altro-
octuluronate-1-P (47) and D-erythro- L-galacto-nonuluronate-1-P (50), and L-threo-L-
galacto-nonuluronate-1-P (51) were shown to be substrates for FBP aldolase. These
compounds showed a range of activity with D-xyluluronate-1-P (34) being 115-fold slower
than FBP (39), while L-glycero-L-galacto-octuluronate-1-P (45) is only 23-fold slower than
FBP (Table S1). Compared to FBP, these are poor substrates, but these experiments
further support the conclusion that the stereochemical arrangements at C3 and C4 are the
same for FBP aldolase and YdjI.
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1
2
2
2
2
2
2
2
2
2
2
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3
4
4
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4
4
4
4
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5
5
5
5
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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9
0
NMR spectroscopy was used to further characterize the reaction product D-
xyluluronate (34) with previously published chemical shift values. D-xyluluronate-1-P (34)
was synthesized by mixing YdjI with DHAP and glyoxylate (3). The resulting product was
dephosphorylated using rSAP and purified by anion exchange chromatography. The 13C
1
3
NMR chemical shifts of the dephosphorylated product are very similar to the published C
chemical shifts for authentic D-xyluluronate synthesized enzymatically by a different route
(
48). The chemical shifts for D-riburonate (the C3 epimer) are reproduced in Table 4 for
comparison.
Table 4. 13C-NMR chemical shifts (ppm).
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Substrate
C1
C2
C3
C4
C5
D-xyluluronatea 66.02 212.37 77.05 73.15 176.90
D-xyluluronateb 65.99 212.38 76.88 73.05 177.13
D-ribuluronateb 66.21 211.13 77.88 74.62 176.43
0
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a
Produced from glyoxylate and DHAP with YdjI after
dephosphorylation . As reported previously (48).
b
Three-Dimensional Structure of YdjI. The crystals utilized in this investigation
contained four subunits in the asymmetric unit, which packed together to form a
tetrameric quaternary structure similar to that observed for tagatose-1,6-bisphosphate
aldolase from E. coli (32). Subsequent gel filtration chromatography confirmed that YdjI
migrates as a tetramer. The final model of YdjI was refined to an overall R-factor of 14.3%
at 1.75 Å resolution. Shown in Figure 6a is a ribbon representation of the tetramer, which
can be envisioned as a dimer of dimers. The total buried surface area between monomers
2
2
A and B is 3500 Å , whereas that between A and C is 1500 Å .
Each subunit is characterized by eight -strands and eleven -helices that form a
classical TIM barrel motif (49). The electron density for Cys234 in each subunit clearly
revealed that it has been oxidized to cysteic acid. It is located on -helix 10 and positioned
near the subunit:subunit interfaces formed by monomers A/B and C/D. In three of the
subunits there were breaks in the electron density corresponding to the polypeptide chain
between a-helices 6 and 7 and between β-strand 6 and α-helix 8. These regions of
flexibility are located at the C-terminal region of the TIM barrel. In monomer B, there was
only a single break in the electron density between a-helices 6 and 7.
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Figure 6. (a) Tetrameric ribbon structure of YdjI. The zinc ion is shown as a gray sphere
(
b) Active site of YdjI. Key residues are labeled and shown as sticks.
As expected for a class II aldolase, in each subunit of the tetramer there is a zinc ion
located at the C-terminal region of the -barrel. Ligation about the zinc is variable in each
subunit as described for other structures (13). In subunit A, the zinc is coordinated by
His82, His178, His206 and two water molecules. This surface-exposed zinc site is typically
found when ligand is bound at the active site, as previously described (13). With respect to
subunit B, however, the zinc is ligated in an octahedral coordination sphere by His82,
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