Sbi00515, a Protein of Unknown Function from Streptomyces bingchenggensis, Highlights the Functional Versatility of the Acetoacetate Decarboxylase Scaffold

Article abstract of DOI:10.1021/acs.biochem.5b00483

The acetoacetate decarboxylase-like superfamily (ADCSF) is a group of ～4000 enzymes that, until recently, was thought to be homogeneous in terms of the reaction catalyzed. Bioinformatic analysis shows that the ADCSF consists of up to seven families that d

Full text of DOI:10.1021/acs.biochem.5b00483

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Article
Sbi00515, a Protein of Unknown Function from
Streptomyces bingchenggensis, Highlights the Functional
Versatility of the Acetoacetate Decarboxylase Scaffold
Lisa S Mueller, Robert W Hoppe, Jenna M Ochsenwald, Robert T Berndt,
Geoffrey B Severin, Alan W. Schwabacher, and Nicholas R. Silvaggi
Biochemistry, Just Accepted Manuscript • Publication Date (Web): 03 Jun 2015
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Sbi00515, a Protein of Unknown Function from
Streptomyces bingchenggensis, Highlights the
Functional Versatility of the Acetoacetate
Decarboxylase Scaffold
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Lisa S. Muellera, Robert W. Hoppea, Jenna M. Ochsenwalda, Robert T. Berndta, Geoffrey B.
Severina, Alan W. Schwabachera, and Nicholas R. Silvaggia*
aDepartment of Chemistry and Biochemistry, University of WisconsinꢀMilwaukee, 3210 North
Cramer Street, Milwaukee, Wisconsin 53211 (USA).
FUNDING STATEMENT: This work was supported by grant MCBꢀ1157392 from the National
Science Foundation, Directorate for Biological Sciences.
KEYWORDS: Enzyme structure and function, aldolase, dehydratase, secondary metabolism,
functional divergence.
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ABBREVIATIONS: ADC, acetoacetate decarboxylase; ADCSF, acetoacetate decarboxylaseꢀlike
superfamily; CaADC, Clostridium acetobutylicum acetoacetate decarboxylase; ShMppR,
Streptomyces hygroscopicus MppR.
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ABSTRACT. The acetoacetate decarboxylaseꢀlike super family (ADCSF) is a group of ~4000
enzymes that, until recently, was thought to be homogenous in terms of the reaction catalyzed.
Bioinformatic analysis shows that the ADCSF consists of up to seven families that differ
primarily in their active site architectures. The soilꢀdwelling bacterium Streptomyces
bingchenggensis BCWꢀ1 produces an ADCSF enzyme of unknown function that has low (~20%)
sequence identity to known acetoacetate decarboxylases (ADCs). This enzyme, Sbi00515,
belongs to the MppRꢀlike family of the ADCSF due to its similarity to the mannopeptimycin
biosynthetic protein MppR from Streptomyces hygroscopicus. Herein we present steady state
kinetic data that show Sbi00515 does not catalyze the decarboxylation of any alphaꢀ or betaꢀketo
acid tested. Rather, we show that Sbi00515 catalyzes the condensation of pyruvate with a
number of aldehydes, followed by dehydration of the presumed aldol intermediate. Thus,
Sbi00515 is a pyruvate aldolaseꢀdehydratase and not an acetoacetate decarboxylase. We have
also determined the Xꢀray crystal structures of Sbi00515 in complexes with formate and
pyruvate. The structures show that the tertiary structure of Sbi00515 is nearly identical to those
of both ADC and MppR. The pyruvate complex is trapped as the Schiff base, providing evidence
that the Schiff base chemistry that drives the acetoacetate decarboxylases has been coꢀopted to
perform a new function, and that this core chemistry may be conserved across the super family.
The structures also suggest possible catalytic roles for several active site residues.
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The acetoacetate decarboxylaseꢀlike superfamily (ADCSF; IPR010451, IPR023375, and
IPR023653) is a largely unstudied group of approximately 4000 proteins. The superfamily
member that has been most thoroughly characterized is the prototypical ADCSF enzyme,
acetoacetate decarboxylase from Clostridium acetobutylicum (CaADC). This enzyme catalyzes
the cleavage of acetoacetate to acetone and CO2 using a Schiff base mechanism involving a
catalytic lysine residue 1-5. To date, most members of the ADCSF are annotated as either
acetoacetate decarboxylases, often on the basis of relatively low sequence identity, or as
unknown/conserved hypothetical proteins.
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Previous studies have shown that, while ADCSF enzymes appear to be structurally
homogenous (e.g. CaADC [3BH2], Chromobacterium violaceum ADC [3BH3], Legionella
pneumophila ADC [3C8W], Methanoculleus marisnigri ADC [3CMB], S. hygroscopicus MppR
[4JM3], and Sbi00515 [4ZBO, this study]), there is some diversity in terms of the substrates used
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and the chemistry catalyzed by members of this superfamily . MppR from S. hygroscopicus
(ShMppR), for example, was observed in Xꢀray diffraction experiments to catalyze the
cyclization of 2ꢀoxoꢀ4ꢀhydroxyꢀ5ꢀguanidinovaleric acid (i.e. "4ꢀhydroxyꢀketoarginine") to give
what would become, after hydrolysis from the enzyme, the keto form of the nonꢀproteinogenic
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amino acid enduracididine . While the precise role of ShMppR in the biosynthesis of
enduracididine remains unclear, it is apparent that very little is known about the degree of
substrate and reaction diversity within the ADCSF.
Not surprisingly, the divergence of ShMppR's catalytic activity from the classical acetoacetate
decarboxylases like CaADC is associated with a number of amino acid substitutions in the active
site. Chief among these are the loss of an Arg side chain proposed to orient the substrate for
decarboxylation in CaADC, loss of the "second Glu" sidechain thought to promote
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decarboxylation in CaADC 3, and an increase in polarity in the neighborhood of the catalytic Lys
in ShMppR. This latter change results in the formation of an αꢀcarboxylateꢀbinding site in MppR
6. Comparative genome analysis of ShMppR and closely related homologs showed that there
exists a subset of ADCSF enzymes that share this same pattern of amino acid substitutions in the
active site. These soꢀcalled MppRꢀlike enzymes comprise one of seven sequence clusters or
families within the ADCSF.
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Herein we describe the structure and biochemical characterization of one of these MppRꢀlike
enzymes, Sbi00515 from Streptomyces bingchenggensis BCWꢀ1. S. bingchenggensis is notable
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not only for the sheer size of its genome—at ~12 million bp (10,106 genes) it is one of the
largest bacterial genomes known—but also for the number of secondary metabolites it produces.
These include the antihelminthic milbamycins A3 and A4 that are used extensively to control
worms in animals, an additional 10 milbemycin analogs 8-11, the polyether antibiotic
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nanchangmycin, the antitumor agents bingchamide A and B , and at least 23 uncharacterized
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nonribosomal peptide, polyketide, or terpene biosynthetic clusters . The gene encoding
Sbi00515 is near one of the uncharacterized polyketide synthase genes, raising the possibility
that Sbi00515 has some role in synthesizing an asꢀyetꢀunknown natural product.
Materials and Methods
Cloning, Expression, and Purification of Sbi00515. The coding sequence of gene product
Sbi00515 was optimized for expression in E. coli and synthesized by GenScript Inc (Piscataway,
NJ). This synthetic gene was subꢀcloned into the pEꢀSUMOkan expression vector (LifeSensors
Inc, Malvern, PA) using primers containing Bsa I and Xba I restriction sites (forward: 5'ꢀ
GGTCTCAAGGTATGAAAGGTTATACGGTTCCGꢀ3';
reverse:
5'ꢀ
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GCTCTAGATCATCATGCCGAATGGTCTGCꢀ3'). The His6ꢀtagged SUMOꢀSbi00515 fusion
protein was expressed from E. coli BL21 Star (DE3) cells (Invitrogen Inc, Carlsbad, CA)
carrying the pEꢀSUMOꢀSbi00515 plasmid. Cultures were grown in LuriaꢀBertani medium with
50 µg/mL kanamycin at 37 °C. At an OD600 of ~0.9, protein expression was induced with 0.4
mM IPTG. The temperature was reduced to 25 °C and the cultures were grown overnight with
shaking at 250 rpm. Cells were harvested by centrifugation, resuspended in 5 mL/g of buffer A
(25 mM TRIS pH 8.0, 300 mM NaCl, 10 mM imidazole) supplemented with 1 mg/mL hen egg
lysozyme (Hampton Research) and frozen at ꢀ20 °C. Cells were lysed by thawing the
resuspended cells for 2 h at RT, then adding 0.1 mg/mL DNAse I (Worthington Biochemical
Corp., Lakewood, NJ). The lysate was clarified by centrifugation at 39,000 x g for 45 minutes
and then applied to a 5 mL HisTrap column (GE Lifesciences, Piscataway, NJ) at a flow rate of 5
mL/min to isolate the His6ꢀSUMOꢀSbi00515 fusion protein. The protein was eluted by a 4ꢀstep
gradient of buffer B (25 mM TRIS pH 8.0, 300 mM NaCl, and 250 mM imidazole; 5, 15, 50, and
100%). The His6ꢀSUMOꢀSbi00515 fusion protein eluted in the third and fourth steps and was
~90 % pure, as judged on coomassieꢀstained SDSꢀPAGE gels. Peak fractions were pooled and
dialyzed overnight against 3.5 L of 25 mM TRIS pH 8.0, 150 mM NaCl in the presence of ~3
µM SUMO protease (LifeSensors Inc). The dialysate was passed through the HisTrap column a
second time to remove the cleaved His6ꢀSUMO tag as well as the protease. The resulting
Sbi00515 preparation was > 95% pure. Selenomethionineꢀlabed Sbi00515 was purified using the
same protocol, except that SelenoMethionine Medium Complete (Molecular Dimensions,
Newmarket, Suffolk, UK) was used as the growth medium rather than LuriaꢀBertani and T7
Express Crystal cells (New England Biolabs, Ipswitch, MA) were used in place of the BL21 Star
(DE3) cells.
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Preparation of Benzylidenepyruvate (1). (E)ꢀ2ꢀoxoꢀ4ꢀphenylbutꢀ3ꢀenoic acid (1) was
prepared by a modification of the technique of Reimer 13. A suspension of benzaldehyde (5.22 g,
49.2 mmol), sodium pyruvate (5.42 g, 49.3 mmol), and KOH (1.41 g, 25.2 mmol) in 15 mL
CH3OH and 15 mL H2O was warmed from < 10 ˚C to 35 ˚C and held at that temperature for 1.5
h, over which time the starting materials dissolved, and then the product crystallized. Filtration,
rinsing with 50 mL cold CH3OH, and drying in vacuo gave 5.25 g of a yellow solid. A solution
of 5.18 g of the yellow solid in 150 mL H2O was filtered, acidified with HCl (2.0 M, 180 mL)
and the resulting precipitate was isolated by filtration, then dried by addition and rotary
evaporation of 50 mL toluene in vacuo to yield 2.91 g of product as an orange solid (34 % yield).
The 1H and 13C NMR spectra of this product were identical to those reported previously 14.
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Steady State Enzyme Kinetics. All 0.5 mL kinetic assays were carried out in triplicate at 25 °C
in 50 mM BisꢀTris, pH 7.0. The initial velocity of Sbi00515ꢀcatalyzed hydrolysis of 1 was
measured directly by monitoring the decrease in absorbance at 330 nm. Likewise, initial
velocities of Sbi00515ꢀcatalyzed condensation of pyruvate with various aldehyde substrates was
also monitored directly. Extinction coefficients and optimum wavelengths for monitoring the
reactions are included in Table 1. The extinction coefficients were determined empirically by
comparing appropriate peaks in the proton NMR spectra of the various enone products produced
in the condensation reactions with the peak for the αꢀproton of tryptophan (added to the NMR
samples as a standard) in order to estimate the concentration of product. The tryptophan stock
concentration was calculated from the absorbance at 280 nm and the known extinction
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coefficient (5540 Mꢀ1 cmꢀ1) . The kcat and Km values were determined from the initial velocity
data using the equation v0 = Vm[A]/(KM + [A]), where [A] is the concentration of the aldehyde or
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benzylidenepyruvate substrate, v0 is the initial velocity, Vm is the maximum velocity, and KM is
the Michaelis constant.
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HPLC Analysis of Sbi00515 Reaction Mixtures. Reaction mixtures (500 ꢁl) containing 12.3
mM 1 in 50mM sodium phosphate, pH 7.4, and 5.7 ꢁM enzyme, as well as authentic standards of
1 and 2 were separated using a 50 x 2.0 mm, 2.5 ꢁm Synergy Polar RP column (Phenomenex) on
an Agilent 1220 HPLC. The gradient ran from 5 to 50 % acetonitrile in 0.1 % TFA/water over 8
minutes at a flow rate of 0.5 mL/min. Analytes were detected by absorbance at 260 nm. All
samples were incubated for 50 min at RT before being passed through 10K MWCO centrifugal
filter devices to remove the enzyme.
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1H NMR Analysis of Sbi00515 Reaction Mixtures. Reaction mixtures (500 ꢁl) containing 2.8
ꢁM Sbi00515 and 12.5 mM 1 or 50 mM 2 with 50 mM pyruvate in 50 mM sodium phosphate
buffer, pH 7.4 (H2O). The reactions were incubated at RT for 80 min (1) or 35 min (2 with
pyruvate), after which the samples were evaporated to dryness in a CentriFan centrifugal
evaporator. The residues were resuspended in 500 ꢁl of D2O. NMR spectra were recorded at RT
on a Bruker DRX500 500 MHz spectrometer equipped with a BBI probe. All data sets consist of
128 scans. Standard samples of 1 (12.5 mM) and of the mixture of 2 with pyruvate (50 mM each)
were treated in the same way as the enzymatic reactions.
Crystallization, Structure Determination, and Model Refinement. Initial crystallization
conditions were identified by screening 45 mg/mL Sbi00515 against the Index HT screen
(Hampton Research). After optimization, diffractionꢀquality crystals were obtained by the
hanging drop vapor diffusion method from 3.5ꢀ3.7 M potassium formate, 2ꢀ3 % polyethylene
glycol monomethylether 2,000 (PEG MME 2K), and 100 mM bisꢀTRIS propane pH 9.0. Drops
contained 1ꢀ2 µl of protein solution at 45 mg/mL and 1 µl of crystallization solution. Crystals
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formed as plates that appeared after 3ꢀ4 days and grew to maximum dimensions of ~800 x 200 x
50 µm. Crystals of selenomethionine (SeMet)ꢀsubstituted Sbi00515 were grown using the same
conditions. Crystals were cryoꢀprotected with Paratone N (Hampton Research) and flashedꢀ
cooled by plunging in liquid nitrogen. Structures of Sbi00515 with pyruvate bound were
obtained by transferring crystals of native Sbi00515 into 30 ꢁL drops of soaking solution
containing 4.0 M potassium formate, 3 % PEG MME 2K, 100 mM bisꢀTRIS propane pH 9.0,
and 30 mM sodium pyruvate. After soaking overnight, crystals were treated with Paratone N and
flashꢀcooled. Xꢀray diffraction data for SeMet Sbi00515 and a higherꢀresolution native data set
were collected at beamline 21ꢀIDꢀD of the Life Science Collaborative Access Team (LSꢀCAT) at
the Advanced Photon Source (APS). The Sbi00515·pyruvate, data set was collected using the
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rotating anode Xꢀray source at Marquette University. Data were processed with HKL2000 or
MOSFLM 17, 18 and SCALA 19 of the CCP4 Program Suite 20.
The structure of Sbi00515 was determined by the singleꢀwavelength anomalous diffraction
(SAD) method using 1.9 Åꢀresolution data collected from a crystal of SeMetꢀsubstituted
Sbi00515 at 0.97921 Å, 4.0 eV above the tabulated Kꢀedge wavelength for Se (0.97950Å). The
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program autoSHARP was used to solve the Se substructure, which contained 12 of the 14 Se
atoms in the asymmetric unit, and calculate densityꢀmodified electron density maps. An initial
model comprising ~75 % of the asymmetric unit contents was built automatically using the
PHENIX package (phenix.autobuild 22, 23). After iterative cycles of manual model building in
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COOT and maximum likelihoodꢀbased refinement using the PHENIX package (phenix.refine
25), ordered solvent molecules were added automatically in phenix.refine and culled manually in
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COOT. Hydrogen atoms were added to the model using phenix.reduce and were included in
the later stages of refinement to improve the stereochemistry of the model. Positions of H atoms
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were refined using the riding model with a global Bꢀfactor. Regions of the model for translationꢀ
librationꢀscrew (TLS) refinement were identified using phenix.find_tls_groups and the TLS
parameters were refined in phenix.refine. Once the refinement converged (e.g. SeMet Sbi00515:
R=0.162, Rfree=0.185) the model was validated using the tools implemented in COOT and
PHENIX 27, 28. Sections of the backbone with missing or uninterpretable electron density were
not included in the final model. Side chains with poor or missing electron density were modeled
in favored rotameric conformations. The Bꢀfactors were allowed to refine without additional
restraints and the occupancies for atoms in those residues without alternate conformations were
held to be 1.0.
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The final, refined model of SeMet Sbi00515, stripped of water molecules and H atoms, and
with all Bꢀfactors set to 20.0 Å2, was used to determine the structures of Sbi00515 “unliganded”
(with formate bound) and with pyruvate bound by difference Fourier. A similar refinement
protocol was used for both models presented here. In the case of the Sbi00515·pyruvate
structure, restraints for the link between Lys122 and pyruvate were generated with phenix.elbow
29. Data collection and model refinement statistics are listed in Table 2. Coordinates and structure
factors have been deposited in the Protein Data Bank (www.rcsb.org) with accession codes
4ZBO and 4ZBT.
Results and Discussion
Sbi00515 was chosen for study based on its sequence similarity to ShMppR (39 % identity)
and its gene context, which suggests a potential role in an uncharacterized polyketide
biosynthetic cluster (Table S1, Supplementary Information). Due to intellectual property
concerns, attempts to obtain samples of live cells or genomic DNA were unsuccessful. As such,
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the precise biological function of Sbi00515 cannot currently be verified. In the absence of this
information, the purpose of this study was to investigate the catalytic capacity of the MppRꢀlike
family specifically, and the ADCSF in general.
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Enzymatic Activity of Sbi00515. Sbi00515 was first assayed for acetoacetate decarboxylase
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activity as described by Highbarger et al . Since Sbi00515 shares the same active site residue
changes relative to CaADC that were observed in ShMppR, it was not surprising that it did not
react with acetoacetate. As ShMppR was shown to react with pyruvate, a number of αꢀketoacids
including glyoxylate, pyruvate, oxaloacetate, and αꢀketoglutarate were also tested as substrates
for Sbi00515. While glyoxylate, pyruvate, and αꢀketoglutarate were observed to form Schiff base
complexes in Xꢀray diffraction experiments (see discussion of pyruvate complex below), and the
enzyme binds pyruvate with a KD of 5.7 ± 0.6 mM (as measured by fluorescence titration), none
of these were altered by the enzyme (e.g. decarboxylated) as judged from HPLC analysis of
reaction mixtures (data not shown).
The similarity of the Sbi00515 structure to that of ShMppR (see below) suggested that it
should be able to react with αꢀketo acids. Sbi00515 was tested for activity with benzylidene
pyruvate (Scheme 1, 1), since any reaction resulting in modification of the double bond (e.g.
hydration) would disrupt the chromophore and be accompanied by a change in the UVꢀvis
absorbance spectrum. UVꢀVis spectroscopy (Figure 1A) showed that incubation of 1 with
Sbi00515 resulted in the timeꢀdependent loss of absorbance at 300 nm and concomitant increase
in absorbance at 254 nm. The structure of 1 and the rise of a species absorbing at 254 nm
suggested that 1 was being hydrolyzed to give benzaldehyde (2) and pyruvate (3; Scheme 1).
That these were products of the reaction was confirmed by HPLC analysis of reaction mixtures
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compared to authentic standards (Figure 1B) and by H NMR spectroscopy (Figure 1C and
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Supplementary Information, Figures S1ꢀS4). These data clearly indicate that Sbi00515 was able
to hydrate the double bond of 1 and catalyze the retroꢀaldol cleavage to give benzaldehyde and
pyruvate. This same reaction has been observed in the transꢀoꢀhydroxybenzylidenepyruvate
hydrataseꢀaldolase from Pseudomonas fluorescens 30, 31, though this enzyme appears, from
secondary structure prediction, to have the TIMꢀbarrel fold typical of Schiff baseꢀforming
aldolases.
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Since these endꢀpoint assays were carried out over relatively long time periods (30 to 120
min), it is possible that the reaction observed was cryptic, and did not represent the physiological
activity of the enzyme. To address this question, both the hydrolysis and condensation reactions
were characterized by steady state enzyme kinetics. Though clearly catalytic, the modest values
of the pseudoꢀsecond order rate constants of 54, 62, and 100 Mꢀ1 secꢀ1 when 1, 2, and 3 are
varied, respectively (Table 1), suggested either that these compounds are nonꢀideal substrates
that participate in a facsimile of the native reaction for this enzyme, or that they induce nonꢀ
physiological chemistry that is only evidence of reaction promiscuity.
In order to clarify this point, a number of additional aldehyde substrates (Scheme 2) were
screened for activity with saturating pyruvate (Table 1). Interestingly, 4ꢀhydroxybenzaldehyde
(4) has a pseudoꢀsecond order rate constant comparable to that of 2, but the KM value is
approximately 200ꢀfold less than that of 2 and kcat is more than 300ꢀfold slower. This may
indicate that Sbi00515 binds 4 more tightly, but in an orientation that is less favorable for
catalysis. Moving from the aryl aldehyde 2 to aliphatic aldehydes 5ꢀ9 results in a marked
improvement in KM with small decreases in kcat, such that the catalytic efficiency improves by as
much as 2 orders of magnitude. There is a clear trend of decreasing KM with increasing chain
length going from 6.6 ± 0.4 mM for 5 to 35.0 ± 2.0 µM for 9. There is no clear relationship
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between substrate size and the kcat values. The complete lack of activity with 7, as judged by
HPLC analysis, shows that the unsaturated bond adjacent to the aldehyde group is required for
the observed catalysis. The aromatic aldehyde 3ꢀ(2ꢀfuryl)acrolein (10) was chosen because it
combines a short, unsaturated alkyl chain with an aromatic group. This substrate was
significantly more efficient than any of the other substrates tested with a kcat value of 22.9 ± 0.8
sꢀ1 and a KM value of 1.2 ± 0.1 mM, for a pseudoꢀsecond order rate constant of 1.9 x 104 Mꢀ1 sꢀ1.
It is interesting to note that the diꢀene substrate 8 reacts 20ꢀfold more slowly than 10 despite the
high similarity of their structures. The ability of the furan ring to accept a hydrogen bond and/or
stack with an aromatic sidechain in the active site may result in 10 binding in a more favorable
orientation for catalysis. Finally, the relatively robust activity with 10 does suggest that the aldol
condensation and dehydration activity of Sbi00515 is real, and not a spurious reaction resulting
from the presence of a reactive enamine in the active site. It should also be noted that the aldol
condensation activity was only observed with pyruvate; other αꢀketo acids that we tested
(glyoxylate, 2ꢀoxobutyrate, oxaloacetate, and αꢀketoglutarate) did not react with any of the
aldehyde substrates used in this study.
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Overall Structure and Comparisons to CaADC and ShMppR. Sbi00515 crystallized in space
group P21212 with 4 molecules in the asymmetric unit (unit cell dimensions a = 157.3, b = 123.5,
c = 53.1, α=β=γ = 90°). The asymmetric unit contents are arranged in a tetramer that matches
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closely the arrangement seen in ShMppR . This tetramer is a dimer of dimers (Figure 2A). The
“dimer interface” is extensive, being comprised of interdigitating loops (Met1ꢀPro21 and
Arg186ꢀGlu203) that bury 2,642 Å2, or 20 % of the protomer surface area. The interface between
the dimers, which we call the “tetramer interface,” is smaller, burying 1621 Å2, or 12 % of the
protomer surface, and is also comprised of two surface loops (Ser68ꢀGln81 and Gln129ꢀGly164).
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The homotetrameric quaternary structure shared by Sbi00515 and ShMppR is markedly different
from that of CaADC, which is a homododecamer. In both cases, the trimerization platform of
CaADC is occluded by the loops of the tetramer interface. This pair of sequence insertions (see
Figure S5) accounts for the difference in quaternary structures between the classical ADCs and
the MppRꢀlike family. Thus far, there is no known functional consequence of the quaternary
structure in ADCSF enzymes.
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The tertiary structure of Sbi00515 is essentially identical to both MppR and CaADC, with root
mean square deviations (RMSD) for all aligned Cα atoms of 1.4 and 1.7 Å, respectively. This
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was not surprising, given that all ADCSF proteins of known structure (PDB IDs 3BH2, 3BH3 ,
4JM3 6, 3C8W (Joint Center for Structural Genomics [JCSG], unpublished), and 3CMB (JCSG,
3
unpublished), share the same βꢀcone or doubleꢀbarrel fold first identified by Ho et al (Figure
2B). In spite of the remarkable similarity of the overall folds, the active sites of Sbi00515 and
CaADC are different. Specifically, as in ShMppR, Arg29, the residue responsible for orienting
the substrate in CaADC and Glu61, thought to electrostatically encourage the carboxylate group
of acetoacetate to leave as CO2, are missing in Sbi00515. These two sequence changes alone
would account for the loss of acetoacetate decarboxylase activity in the MppRꢀlike enzymes.
The other major difference between CaADC and the MppRꢀlike enzymes is the presence of the
very polar "carboxylateꢀbinding site" adjacent to the catalytic lysine residue (Lys122 in
Sbi00515). This site is marked in the “unliganded” structure of Sbi00515 by a bound formate ion
from the crystallization solution (Figures 3A and 4A). The ion is held by hydrogen bonding
interactions between O1 of formate and the hydroxyl group of Tyr24 (2.6 Å) or the guanidinium
group of Arg114 (2.7 Å), and between O2 of formate and the amide nitrogen atom of Gln118
(2.9 Å) or the amino group of Lys122 (2.7 Å). Two water molecules are also bound in close
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proximity to the catalytic Lys122 and the bound formate ion. One, labeled “1” in Figure 4A, is
bridging Tyr82 and Glu84 (equidistant at 2.7 Å), while the other, labeled “2” is held by
interactions to water “1” (2.9 Å) and the hydroxyl group of Tyr252 (2.6 Å). The position of
water molecule “1,” only 3.3 Å from the amino group of Lys122, suggests that Tyr82 and Glu84
might both participate in the Schiff base chemistry of Lys122.
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The active site of Sbi00515 does differ subtly from that of ShMppR, which explains the fact
that the latter is able to catalyze the condensation of imidazoleꢀ4ꢀcarboxaldehyde and pyruvate 6.
In Sbi00515, Tyr252 replaces the Glu283 of MppR that is thought to help orient the guanidinium
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group of a hydroxylated arginine derivative for cyclization . In addition, Tyr82 is a
phenylalanine residue in MppR (Phe116), and Tyr24 of Sbi00515 is also a phenylalanine in
ShMppR (Phe58). All of the other residues are identical (Figure 5A). Tyr24 of Sbi00515 is
especially interesting, since it is a component of the carboxylateꢀbinding site not present in
ShMppR and alters the conformation of the Schiff base complex between pyruvate and Lys122.
Schiff Base Complex with Pyruvate. Crystals of Sbi00515 were soaked in a solution containing
30 mM sodium pyruvate, and the resulting covalent imine complex was trapped
crystallographically (Figures 3B and 4B). The carboxylate moiety of pyruvate is bound in the
carboxylateꢀbinding site of the enzyme, making hydrogen bonding interactions to the hydroxyl
groups of Tyr24 and Tyr82 (2.6 and 3.2 Å, respectively) and the amide nitrogen atom of Gln118
(2.8 Å), as well as a potential salt bridge with Arg114 (bidentate at 3.0 Å). Binding of pyruvate
does not impact the positions of the side chains comprising the carboxylateꢀbinding site (Tyr24,
Arg114, and Gln 118; Figure 5B), indicating that this site is preꢀconfigured to bind αꢀketo acids.
This may explain, at least in part, why the Schiff base complex is sufficiently stable to be
observed in the crystal structure. The residues closer to the catalytic center, Tyr82, Glu84, and
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Val106 experience small, but significant and concerted, changes in their positions. Tyr82 swings
toward the catalytic lysine moving the hydroxyl group by 0.6 Å, while Glu84 swings away from
Lys122 shifting its carboxylate group by 1.4 Å. The distance between the nearest carboxylate O
atom of Glu84 and the pyruvate methyl group is 3.9 Å. Given this arrangement, it is plausible to
suggest that Glu84 might act as a general base catalyst to promote formation of the reactive
enamine form of the Schiff base. Lastly, although it is some distance from the Lys122ꢀGlu84
dyad, Tyr252 also experiences a 0.6 Å shift of its hydroxyl group toward Lys122, likely due to a
rearrangement of the water structure around the catalytic center that occurs upon formation of the
Schiff base (Figure 5B). In the pyruvateꢀbound structure, the water bridging Tyr82 and Glu84 is
displaced (water “1” in Figure 4A) and two additional water molecules are recruited (labeled “3”
and “4” in Figure 5B) that create a chain connecting Glu84, Gln118, and Tyr252. The position of
water molecule “2” moves along with Tyr252 by 0.8 Å, placing it 3.6 Å away from the pyruvate
methyl group. The position of this water is instructive, because when the incoming aldehyde
substrate is placed so that the aldehyde O atom overlays water “2” and the re face of the carbonyl
faces the pyruvate methyl group, the distance between the methyl and carbonyl carbons is
approximately 3 Å.
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It was somewhat surprising to find that although pyruvate occupies the same binding site in
Sbi00515 and ShMppR, the conformations of the Schiff base complexes are different. In
ShMppR (PDB ID 4JMC), the pyruvate carboxylate group is nearly coplanar with the
guanidinium of Arg148, making a clear salt bridge interaction. In Sbi00515, the substitution of
Tyr24 for Phe58 of ShMppR results in the carboxylateꢀbinding site Arg and Gln residues taking
different conformations. As a result, the pyruvate carboxylate in the Sbi00515·pyruvate complex
is rotated by ~30 ° relative to the ShMppR·pyruvate structure. The rotation of the carboxylate
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weakens or eliminates the salt bridge to Arg114 and creates a new interaction with Tyr24. This
change also forces the carboxylate to nearly eclipse the methyl group (O2ꢀC1ꢀC2ꢀC3 torsion
angle is 14 °). While it is possible that this nearly eclipsed conformation just happens to be the
lowestꢀenergy conformation in this complex, the strain inherent in this arrangement, the 2.8 Å
distance between the carboxylate oxygen and the methyl carbon, and the potential loss of the salt
bridge raise the possibility of substrateꢀassisted catalysis. It is possible that the carboxylate could
abstract a proton from the former pyruvate methyl carbon during the transition from the
presumed aldol intermediate to the unsaturated product. A substrate carboxylate is proposed to
act as a general acid/base catalyst in sialic acid aldolase 32.
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Conclusion. The steady state kinetic data presented here show that Sbi00515, a protein of
unknown function from S. bingchenggensis, has in vitro pyruvate aldolaseꢀdehydratase activity,
and is not an acetoacetate decarboxylase as it is currently annotated. This is the first Schiff baseꢀ
forming aldolase known to use a fold other than the TIMꢀbarrel. Given the proximity of the gene
encoding Sbi00515 to an uncharacterized polyketide biosynthetic cluster and the strong
preference for an unsaturated aldehyde substrate, it is tempting to speculate that Sbi00515 might
synthesize an unusual starter or extender unit for the polyketide. However, more detailed genetic
and biological studies must be done before anything substantive can be concluded in this regard.
The structures of the enzyme in complexes with formate and pyruvate point out possible roles of
several active site amino acids. Both Tyr82 and GluE84 are near enough to the catalytic lysine
residue to make contacts to the presumed hemiaminal intermediate during formation and
breakdown of the Schiff base. Since only Glu84 is in a position to reach both Lys122 and the
methyl group of pyruvate, and Tyr82 is not absolutely conserved in MppRꢀlike enzymes, Glu84
is the most likely candidate for the general acid/base catalyst. The position of the water molecule
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held by Y252 in the presence and absence of pyruvate implies that this residue might orient the
incoming aldehyde substrate for attack by the enamine nucleophile. Kinetic characterization of
active site variants is needed to explore the roles of these residues more deeply. In all, these
experiments show that there is significant reaction diversity not only in the ADCSF, but also
within the MppRꢀlike family itself.
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Figure 1. (A) The Sbi00515ꢀcatalyzed cleavage of 1 observed by UVꢀvis spectroscopy. The
spectra are separated by 4.5 min, and thus cover a 90 minute period. The 500 µl reaction
contained 100 mM 1 and 12.8 µM Sbi00515 in 10 mM MES, pH 6.0. The reaction was initiated
by the addition of enzyme. (B) HPLC analysis of reaction mixtures and authentic benzaldehyde
(2) suggest that Sbi00515 catalyzes the hydrolysis of 1 to give 2 and pyruvate (3). Starting
material (1, blue trace) and benzaldehyde, the putative product (2, red trace), are very well
resolved, eluting at 6.3 and 3.9 min, respectively. A sample containing 12.3 mM 1 and 5.7 µM
Sbi00515 (green trace) clearly shows a loss of starting material with corresponding accumulation
of a product that also elutes at 3.9 min under these conditions. (C) Proton NMR spectra of
reaction mixtures and standards confirm that the products of the reaction between Sbi00515 and
1 are 2 and pyruvate. The authentic starting material (1, red trace) gives a spectrum that is
distinct from that of benzaldehyde (cyan trace). The doublet at 6.8 ppm is characteristic of the
unsaturated bond. The methyl protons of pyruvate give a singlet at 2.25 ppm (cyan trace). After
incubating with the enzyme, pyruvate is clearly present (green trace). Given 50 mM pyruvate,
the reaction can be run in reverse (purple trace), where the doublet at 6.8 ppm confirms the
production of 1. Together, these data confirm that Sbi00515 has in vitro aldolaseꢀdehydratase
activity. The complete NMR spectra are provided in the Supplementary Information as figures
S1ꢀS4.
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Figure 2. Rendering of the solvent accessible surface of Sbi00515 (A) showing the extensive
tetramer interface formed by the long, interdigitating loops (top). This interface is repeated on
the back side with the orange and pale yellow protomers. The pair of dimers is held together
(bottom) by short loops that appear to “clip” the two dimers together to form the tetramer. The
ribbon diagram showing a single protomer (B) is oriented similarly to the dark blue protomer in
panel A. The main barrel of the βꢀcone fold is shown in pale blue, with the “side barrel” in green.
The dimer interface loops are colored yellow and the loops forming the interface between the
dimers are colored orange. The section of the polypeptide that is colored red is the “active site
loop” that may control access to the active site. The catalytic Lys122 is shown as a ballꢀandꢀstick
representation with purple carbon atoms.
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Figure 3. Stereoview of the active site in the “unliganded” Sbi00515 structure showing
potassium formate from the crystallization solution bound (A; refer to Figure 4 for relevant
hydrogen bonding interactions and their distances). The position of this formate ion shows that
the carboxylateꢀbinding site identified in S. hygroscopicus MppR is retained Sbi00515. The
lower water molecule in this view (labeled “1” in Figure 4) bridges Tyr82 and Glu84, suggesting
that both residues could participate in the Schiff base chemistry. The upper water molecule (“2”
in Figure 4) is held in place by interactions with Tyr252, which is the major difference between
the Sbi00515 and ShMppR active sites. The Sb00515·Pyruvate Schiff base complex (B) shows
pyruvate covalently bound to the enzyme as indicated by continuous electron density between
Lys122 and the alpha carbon of pyruvate. The side chain of pyruvate is too short to
unequivocally differentiate between the imine and enamine forms of the Schiff base. Note that
water “1” has been displaced, water “2” remains in a similar position, and two new water
molecules have been recruited so that Glu84, Gln118, and Tyr252 are all linked by an extensive
hydrogen bonding network. In both panels, the experimental 2|Fo|ꢀ|Fc| electron density map
(magenta) and the simulated annealing composite omit 2|Fo|ꢀ|Fc| electron density map (green),
both contoured at 1.0 σ, are drawn with a 2.0 Å radius around each atom of Lys122, formate or
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pyruvate, and select water molecules. This figure and subsequent figures showing
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crystallographic structures were rendered with the POVSCRIPT+
MOLSCRIPT 34 and POVRAY.
modification of
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Figure 4. Schematic views of the Sbi00515·formate complex (A) and the Sbi00515·pyruvate
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complex (B) showing potential hydrogen bonding interactions and their distances (in Å).
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Figure 5. Stereo view showing the complex of Sbi00515 and formate (pale blue model with
purple formate ion) overlayed on the enzyme with pyruvate bound (dark blue model; A). Note
the subtle changes in position that occur for residues Tyr82, Glu84, and Tyr252, while the
residues comprising the carboxylateꢀbinding site are essentially unperturbed. In (B) the
MppR·pyruvate complex (green model) has been overlayed on the Sbi00515·pyruvate complex
(dark blue model). In spite of the high degree of structural similarity (1.7 Å RMSD for all
aligned Cα atoms), the arrangement of residues in the carboxylateꢀbinding site is subtly different
due to the addition of Tyr24 in Sbi00515. This change results in Sbi00515 binding the pyruvate
carboxylate with a monodentate interaction to Arg114 and an additional hydrogen bonding
interaction to Tyr24, which holds the carboxylate in plane with the pyruvate methyl group (2.8 Å
from CH3 to carboxylate O). This change, coupled with the Glu > Tyr change at position 283/252
may, in part, account for the difference in catalytic activity between Sbi00515 and MppR.
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Scheme 1.
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Scheme 2.
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Table 1. Steady state kinetic parameters measured for the reaction of 2.0 ꢁM Sbi00515 with the
enone substrate 1 in the absence of pyruvate, saturating concentrations of pyruvate (50 mM) and
various aldehyde substrates (see Schemes 1 and 2), subꢀsaturating concentrations of 2 (50 mM)
and pyruvate (3), or saturating concentrations of 10 (10 mM) and pyruvate. As described in the
text, the poor binding and limited solubility of 2 made it impossible to fully saturate the enzyme.
The kinetic parameters apply to the substrate in column 1. The extinction coefficients given are
for benzylideneꢀpyruvate itself (row 1) and the condensation products of pyruvate with the
aldehyde substrates tested (remaining rows).
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kcat
(sꢀ1)
KM
kcat/KM
(Mꢀ1 sꢀ1)
Product
λmon (nm) ε (Mꢀ1 cmꢀ1)
Substrate
Benzylideneꢀ
pyruvate (1)
(mM)
1.2 ± 0.20
3.2 ± 0.16
22.3 ± 7.0
51.5 ± 6.1
5.4 x 101
6.2 x 101
2.5 x 101
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360
24,159
11,426
1,217
Benzaldehyde (2)
4ꢀHydroxyꢀ
benzaldehyde (4)
0.01 ± 0.001 0.4 ± 0.05
transꢀ2ꢀPentenal (5)
transꢀ2ꢀHexenal (6)
Hexanal (7)
1.2 ± 0.05
12.9 ± 0.6
6.6 ± 0.4
1.8 ± 0.1
1.8 x 102
7.2 x 103
—
290
290
—
26,021
8,576
—
No Reaction —
2,4ꢀHeptadienal (8)
0.90 ± 0.04 0.3 ± 0.02
3.0 x 103
360
2,432
3,7ꢀDimethylꢀ2,6ꢀ
octadienal (citral, 9)
0.28 ± 0.01 0.04 ± 0.002 7.0 x 103
330
390
3,393
transꢀ3ꢀ(2ꢀ
furyl)acrolein (10)
22.9 ± 0.8
1.2 ± 0.08
1.9 x 104
10,062
Pyruvate (3)a
Pyruvate (3)b
1.75 ± 0.07 17.2 ± 2.8
22.5 ± 1.2 11.8 ± 1.6
1.0 x 102
1.9 x 103
300
390
—
—
a Due to the high KM value for 2, reactions were not saturated with respect to this substrate.
b Reaction kinetics of pyruvate with 10 were nearly saturated (the concentration of 10 was
~10ꢀfold greater than the measured KM value).
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Table 2. Crystallographic data collection and model refinement statistics.
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SeMet Sbi00515
Native Sbi00515
Sbi00515·Pyruvate
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Resolution (Å)
(last shell)a
46.5ꢀ1.9
(1.97ꢀ1.90)
41.58ꢀ1.4
(1.45ꢀ1.40)
39.9ꢀ1.8
(1.86ꢀ1.80)
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0.97921
0.97896
1.54180
Wavelength (Å)
No. of reflections
Observed
1131019 (96476)
82988 (8183)
100.0 (100.0)
0.145 (0.456)
13.6 (11.8)
22.3 (5.9)
1626413 (91229)
207128 (20393)
99.9 (99.5)
0.071 (0.560)
7.9 (4.5)
285228 (8521)
92300 (6104)
95.4 (64.0)
0.102 (0.334)
3.1 (1.4)
Unique
Completeness (%)a
R
merge (%)a,b
Multiplicity
<I/σ(I)>a
Figureꢀofꢀmerit (Acentric)
Phasing power
20.5 (2.2)
12.3 (1.7)
0.283
1.102
Model Refinement Statistics
Reflections in work set
Reflections in test set
Rcryst (Rfree)
No. of residues
No. of solvent atoms
Number of TLS groups
Average Bꢀfactor (Å2) c
Protein atoms
Ligand atoms
Solvent
RMS deviations
Bond lengths (Å)
Bond angles (°)
Coordinate error (Å)
Ramachandran statistics
(favored/allowed/outliers)
78722
4185
0.162 (0.185)
1023
1166
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207021
2960
0.154 (0.176)
1034
1385
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92221
4631
0.163 (0.205)
1028
1289
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15.9
35.6
26.9
21.0
33.9
34.1
21.4
31.9
31.3
0.005
0.979
0.30
0.009
1.303
0.12
0.011
1.309
0.18
98.2 / 1.8 / 0
97.7 / 2.3 / 0
97.2 / 2.8 / 0
a Values in parentheses apply to the highꢀresolution shell indicated in the resolution row.
b R = Σ(||Fobs|ꢀscale*|Fcalc||) / Σ |Fobs|.
c Isotropic equivalent B factors, including contribution from TLS refinement.
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ASSOCIATED CONTENT
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Supporting Information. Table S1 gives the genomic context of the gene encoding Sbi00515.
Figures S1ꢀS4 give the full NMR spectra of the substrates and reaction products described in the
text. Figure S5 shows an alignment of Sbi00515, ShMppR and CaADC. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed: Nicholas R. Silvaggi, Ph.D.: 3210 North
Cramer Street, Milwaukee, WI 53211, 414ꢀ229ꢀ2647, silvaggi@uwm.edu.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S.
Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported
by the U.S. DOE under Contract No. DEꢀAC02ꢀ06CH11357. Use of the LSꢀCAT Sector 21 was
supported by the Michigan Economic Development Corporation and the Michigan Technology
TriꢀCorridor (Grant 085P1000817). This work was supported by grant MCBꢀ1157392 from the
National Science Foundation, Directorate for Biological Sciences.
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REFERENCES.
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7266ꢀ7269.
[2] Highbarger, L. A., Gerlt, J. A., and Kenyon, G. L. (1996) Mechanism of the reaction
catalyzed by acetoacetate decarboxylase. Importance of lysine 116 in determining the pKa of
activeꢀsite lysine 115, Biochem 35, 41ꢀ46.
[3] Ho, M. C., Menetret, J. F., Tsuruta, H., and Allen, K. N. (2009) The origin of the
electrostatic perturbation in acetoacetate decarboxylase, Nature 459, 393ꢀ397.
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[6] Burroughs, A. M., Hoppe, R. W., Goebel, N. C., Sayyed, B. H., Voegtline, T. J.,
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