Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis

Article abstract of DOI:10.1074/jbc.M112.351809

Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia- lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of D- and L-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 A resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with DL-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp¹²⁰ and Lys⁷⁷ is suggested.

Full text of DOI:10.1074/jbc.M112.351809

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20369–20381, June 8, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Crystal Structure of Escherichia coli Diaminopropionate
Ammonia-lyase Reveals Mechanism of Enzyme Activation
□
S
and Catalysis*
Received for publication, February 14, 2012, and in revised form, April 12, 2012 Published, JBC Papers in Press, April 13, 2012, DOI 10.1074/jbc.M112.351809
Shveta Bisht‡1, Venkatesan Rajaram‡, Sakshibeedu R. Bharath‡1, Josyula Nitya Kalyani§, Farida Khan§,
Appaji N. Rao§, Handanahal S. Savithri§, and Mathur R. N. Murthy‡2
From the ‡Molecular Biophysics Unit, §Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
Background: DAPAL is a novel PLP-dependent enzyme involved in the degradation of DAP, a nonstandard amino acid.
Results: Striking structural differences were observed between apo- and holo-EcDAPAL, and structure with a PLP-aminoac-
rylate intermediate bound at the active site was obtained.
Conclusion: Enzyme undergoes apo to holo structural transitions and follows a two-base mechanism of catalysis.
Significance: The results provide insights into the catalytic mechanism of DAPAL.
Diaminopropionate (DAP)3 is a nonstandard amino acid
found in both prokaryotes and higher organisms (1–3). In pro-
karyotes, DAP is an important intermediate in the synthesis of
nonribosomal peptides exhibiting antibiotic properties such as
viomycin (3), capreomycin (4), and zwittermicin (5). It has been
demonstrated that consumption of large quantities of a
Pyridoxal 5ꢀ-phosphate (PLP)-dependent enzymes utilize the
unique chemistry of a pyridine ring to carry out diverse reac-
tions involving amino acids. Diaminopropionate (DAP) ammo-
nia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that
catalyzes the degradation of D- and L-forms of DAP to pyruvate
and ammonia. Here, we report the first crystal structure of
DAPAL from Escherichia coli (EcDAPAL) in tetragonal and drought-resistant legume Lathyrus sativus leads to neurolathy-
rism, a degenerative disease caused by the neurotoxin ␤-N-
oxalyl-L-␣,␤-diaminopropionate (6, 7). L-DAP is the immediate
precursor in the synthesis of the neurotoxin (8). High levels of
D-DAP have been found in the gut of Bombyx larvae (2), which
reduce drastically in the pupal stage (9).
monoclinic forms at 2.0 and 2.2 Å resolutions, respectively.
Structures of EcDAPAL soaked with substrates were also deter-
mined. EcDAPAL has a typical fold type II PLP-dependent
enzyme topology consisting of a large and a small domain with
the active site at the interface of the two domains. The enzyme is
a homodimer with a unique biological interface not observed
earlier. Structure of the enzyme in the tetragonal form had PLP
bound at the active site, whereas the monoclinic structure was in
the apo-form. Analysis of the apo and holo structures revealed
that the region around the active site undergoes transition from
a disordered to ordered state and assumes a conformation suit-
able for catalysis only upon PLP binding. A novel disulfide was
found to occur near a channel that is likely to regulate entry of
ligands to the active site. EcDAPAL soaked with DL-DAP
revealed density at the active site appropriate for the reaction
intermediate aminoacrylate, which is consistent with the obser-
vation that EcDAPAL has low activity under crystallization con-
ditions. Based on the analysis of the structure and results of
site-directed mutagenesis, a two-base mechanism of catalysis
involving Asp120 and Lys77 is suggested.
Pyridoxal 5Ј-phosphate (PLP)-dependent enzymes are cru-
cial for the metabolism of amino acids and have been exten-
sively investigated in terms of their structure and function.
These enzymes carry out a variety of reactions, such as
transamination, racemization, deamination, decarboxylation,
elimination, and replacement, using PLP as an electron sink for
stabilization of reaction intermediates. They have been classi-
fied into four major folds (I–IV) based on their structure and
sequence similarities (10, 11). Diaminopropionate ammonia-
lyase (DAPAL) (EC 4.3.1.15), a prokaryotic enzyme, has been
classified as a fold type II PLP-dependent enzyme, although it
shares low sequence identity (18–23%) with other well studied
fold type II enzymes. DAPAL catalyzes the conversion of both
D- and L-isoforms of DAP to pyruvate and ammonia (Fig. 1)
(12). The only other substrate it can degrade, although poorly, is
serine. In eukaryotes, which appear to lack DAPAL, cystathio-
nase has been shown to carry out degradation of DAP, albeit at
a lower rate compared with that of its cognate substrate cysta-
thionine (13). Biochemical studies on the catalytic properties of
DAPAL from Pseudomonas sp. (12), Salmonella typhimurium
(14, 15), and Escherichia coli (14, 16) have been reported in the
literature. However, in the absence of its crystal structure, these
* This work was supported in part by the Department of Biotechnology, the
Department of Science and Technology, and J. C. Bose fellowship, Govern-
ment of India.
□S
This article contains supplemental Methods, Results, and Figs. S1 and S2.
The atomic coordinates and structure factors (codes 4D9G, 4D9I, 4D9K, 4D9M,
and 4D9N) have been deposited in the Protein Data Bank, Research Collabo-
ratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
3 The abbreviations used are: DAP, diaminopropionate; PLP, pyridoxal
5Ј-phosphate; DAPAL, DAPammonia lyase; EcDAPAL, E. coli DAPAL; SeMet,
selenomethionine; DSD, D-serine deaminase; LSD, L-serine dehydratase;
SR, serine racemase; r.m.s.d., root mean square deviation; PDB, Protein
Data Bank.
1 Recipient research fellowships from the Council for Scientific and Industrial
Research, Government of India.
2 To whom correspondence should be addressed. Tel.: 91-80-2293l-2458; Fax:
91-80-2360-0535; E-mail: mrn@mbu.iisc.ernet.in.
JUNE 8, 2012•VOLUME 287•NUMBER 24
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Structure and Function of Diaminopropionate Ammonia-lyase
program HySS (21) located 36 tentative Se positions corre-
sponding to a dimer in the asymmetric unit, out of which 26
were accepted for phase calculation in PHASER (22). The figure
of merit based on these sites was 0.54. The map was improved
by density modification and NCS averaging using Resolve of
Phenix suite. The improved map was used for chain tracing.
Three noncontiguous segments consisting of a total of 758 res-
idues (698 with side chains) were identified by Resolve. The
model and the map had a correlation coefficient of 0.81 and
FIGURE 1. Reaction catalyzed by EcDAPAL.
investigations did not reveal the structural basis of its substrate
and reaction specificity and mechanism of catalysis.
R
work and Rfree of 25.0 and 28.0%, respectively. The model was
Three-dimensional structures of DAPAL from E. coli
(EcDAPAL) in holo- and apo-forms and structures of the com-
plexes of the enzyme with substrates are described for the first
time in this paper. The structures and observations on ligand
binding and catalysis provide insights into the mode of activa-
tion of enzyme upon PLP binding and mechanism of efficient
degradation of both D and L stereo forms of the substrate.
improved by several rounds of refinement using REFMAC5
(23) and manual model building using COOT (24) of CCP4
suite (25). Solvent molecules were identified by the automatic
water-picking algorithm of COOT. The positions of these auto-
matically picked waters were manually checked, and a few more
waters were identified on the basis of electron density con-
toured at 1.0␴ in the 2Fo Ϫ Fc map and 3.0␴ in the Fo Ϫ Fc map.
In the final stages of refinement, inspection of the difference (Fo
EXPERIMENTAL PROCEDURES
Cloning, Overexpression, and Purification—Cloning, expres- Ϫ Fc) map showed strong positive density into which a model of
sion, and purification of EcDAPAL have been described previ- the co-factor PLP could be built. The final structure of SeMet
ously (14). EcDAPAL polypeptide consists of 398 residues with EcDAPAL modeled in tetragonal crystal form contains two
a molecular mass of 43.3 kDa. Selenomethionine (SeMet)-in- polypeptide chains, two PLP molecules (bound to Lys77), two
corporated EcDAPAL was prepared by the metabolic inhibition Tris ions, and 242 water molecules.
method (17). Details are described in supplemental Methods.
Other structures were determined using the SeMet
Crystallization and Data Collection—Crystals of EcDAPAL EcDAPAL as the phasing model. The refinement statistics for
suitable for structural studies were obtained as reported previ- all structures are given in Table 1. The first (Met1) and the last
ously (18). Two crystal forms were obtained under similar con- (Pro398) residues of the polypeptide were disordered in most of
ditions containing 20% PEG 3350, 70 mM MgCl2, 50 mM Li2SO4, the structures. Crystals soaked with DL-DAP, pH 6.5, and L-Ser
ϩ
Ϫ
6 mM Na C2H5COO , 40 mM Tris-HCl, pH 8.3, and varying did not have any extra density corresponding to bound sub-
concentrations of ␤-mercaptoethanol (0–5 mM). Plate-like strate or reaction intermediate. These structures were similar
crystals belonged to the tetragonal (P43212) space group, to EcDAPAL structure obtained without soaking with sub-
although the rod-shaped crystals were of the monoclinic (P21) strates and therefore have not been deposited in the Protein
space group. Only tetragonal crystals appeared when excess Data Bank. Residue Cys48 of all protomers in monoclinic form
PLP (50–100 ␮M) was added to the crystallization drops. SeMet and SeMet derivative structure in the tetragonal form has been
EcDAPAL also crystallized in the same two forms under similar modified by ␤-mercaptoethanol to hydroxyethylthiocysteine.
conditions. Crystals were briefly soaked in a cryo-protectant This cysteine is a surface residue, and its chemical modification
solution (crystallization condition with additional PEG 3350 up is unlikely to have any implication on the activity of the protein.
to 30%), mounted on a cryo-loop and frozen in liquid nitrogen Similar modification of surface-exposed residues by ␤-mercap-
for x-ray diffraction data collection.
toethanol has been observed in 165 other protein structures
Diffraction data were collected for native and SeMet deposited in the PDB.
EcDAPAL in the tetragonal form, native monoclinic crystals,
Structure Analysis—The geometries of the final models were
tetragonal crystals soaked in crystallization solution containing checked using PROCHECK (26). Structural superposition and
10 mM D-Ser, L-Ser, or DL-DAP, and tetragonal crystals soaked root mean square deviation (r.m.s.d.) calculations between the
with DL-DAP at a lower pH (6.5). Details are described in sup- subunits of EcDAPAL were carried out using the program
plemental Methods. All datasets were processed and scaled ALIGN (27) and SSM superpose (28) feature of COOT. DALI
using the programs DENZO and SCALEPACK of the HKL suite server was used for the identification of structural homologs
(19). Both tetragonal and monoclinic crystals showed large (29) in the PDB. Average B-factors for protein atoms, water
batch to batch variations in the unit cell parameters (b in tet- molecules, and ligands were calculated using the BAVERAGE
ragonal and both b and c in monoclinic), due to which initial program of the CCP4 suite. PISA was used for analysis of inter-
attempts to determine structure by isomorphous replacement faces and identification of interface residues (30). Figures were
method were not successful. These variations were found to prepared using the programs PyMOL (31) and ChemDraw.
result from weak packing of molecules along the variable cell
Site-directed Mutagenesis—Single site mutants of EcDAPAL
edges as discussed in the supplemental Results and supplemen- targeting residues Lys77, Asp120, and Asp189 were constructed
tal Fig. S1.
by site-directed mutagenesis (32). EcDAPAL cloned in pRSET
Structure Determination and Refinement—The structure C vector was used as the template for generation of mutants.
was determined at 2.5 Å using the data collected from a SeMet The primers used for generating mutants are listed in supple-
EcDAPAL crystal by single wavelength anomalous dispersion mental Methods. All the mutants were confirmed by
method using the Autosol program of Phenix suite (20). The sequencing.
20370 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure and Function of Diaminopropionate Ammonia-lyase
TABLE 1
Data collection and refinement statistics
Values in parentheses refer to the highest resolution shell.
Dataset name
Wavelength
SeMet
0.98
Native-P4
1.0
Native-P2
0.93
DL-DAP
1.54
D-Ser
1.54
Cell parameters
a
b
c
85.64 Å
85.53 Å
85.78 Å
85.51 Å
85.61 Å
85.64 Å
85.53 Å
94.15 Å
85.51 Å
85.61 Å
207.27 Å
90, 90, 90°
P43212
207.27 Å
90, 90, 90°
P43212
94.69 Å
205.08 Å
90, 90, 90°
P43212
207.65 Å
90, 90, 90°
P43212
␣, ␤, ␥ (degrees)
90, 110.01, 90°
P21
Space group
Resolution range
50.0–2.45
(2.49–2.45 Å)
7.5% (19.9%)
53,936
20.0–2.00
(2.03–2.00 Å)
6.6% (37.5%)
52,570
50.0–2.20
(2.28–2.20 Å)
7.8% (35.7%)
71,324
50.0–2.50
(2.54–2.50 Å)
8.1% (41.8%)
26,585
50.0–2.50
(2.54–2.50 Å)
11.4% (38.9%)
27,635
a
Rmerge
Unique reflections
b
͗I͘/͗ ␴I͘
49.3 (14.0)
99.7% (93.4%)
15.4 (13.1)
19.3%
22.2 (2.2)
99.2% (93.6%)
5.0 (3.0)
19.2%
18.9 (2.5)
98.3% (97.5%)
3.6 (3.4)
21.3%
29.2 (3.9)
97.4% (98.1%)
6.1 (6.1)
21.8%
31.2 (3.6)
99.8% (97.3%)
9.5 (5.0)
21.7%
Completeness
Redundancy
c
Rwork
d
Rfree
24.1%
23.3%
26.1%
26.7%
26.7%
r.m.s.d. bond length
r.m.s.d. bond angle
0.008 Å
0.009 Å
0.005 Å
0.006 Å
0.005 Å
1.169°
1.222°
1.022°
0.990°
0.905°
Ramachandran plot
Favored
92.3%
7.3%
0.1%
0.3%
9
91.1%
8.4%
0.1%
0.3%
4
91.1%
8.1%
0.4%
0.4%
8
90.8%
8.7%
0.2%
0.3%
4
91.1%
8.4%
0.1%
0.3%
4
Additionally allowed
Generously allowed
Outliers
TLS groups per chain
Average B-factor
Protein atoms
27.43 Å2
11.44 Å2
17.34 Å2
37.62 Å2
32.10 Å2
29.21 Å2
44.10 Å2
33.10 Å2
57.89 Å2
54.01 Å2
33.79 Å2
42.03 Å2
44.28 Å2
25.28 Å2
34.55 Å2
Water atoms
Other hetero-atoms
a
R
merge ϭ (⌺hkl⌺i͉Ii(hkl) Ϫ ͗I(hkl)͉͘)/⌺hkl⌺Ii(hkl), where Ii(hkl) is the intensity of the ith measurement of reflection (hkl) and ͗I(hkl)͘ is its mean intensity.
b I is the integrated intensity, and ␴(I) is the estimated standard deviation of that intensity.
c Rwork ϭ (⌺hkl͉Fo Ϫ Fc͉)/⌺hklFo, where Fo and Fc are the observed and calculated structure factors.
d Rfree is calculated as for Rwork but from a randomly selected subset of the data (5%), which were excluded from the refinement process.
Activity Assay and Spectral Studies—Activity measurements S8–S11) mixed and twisted ␤-sheet surrounded by 11 helices
of EcDAPAL and its mutants were carried out by a coupled
enzyme spectrophotometric method by monitoring the
decrease in absorption at 340 nm due to consumption of
NADH (14), as described in supplemental Methods. All assays
were carried out in 50 mM potassium phosphate buffer contain-
ing 10 ␮M PLP at pH 7.5. The visible absorbance spectrum of
EcDAPAL (1 mg mlϪ1) and mutants in the range of 300–550
nm in potassium phosphate buffer or crystallization solution
was recorded using a Jasco UV-Visible V-630 spectrophotom-
eter. Spectra were also recorded as a function of time upon
addition of substrates (10 mM).
(H1–H3 and H10–H17). The central five strands (S3 and
S8–S11) are parallel, whereas the strands at the edges (S1 and
S2) are antiparallel and are at an angle of 70° to each other due
to twisting of the sheet. The small domain is made up of a
four-stranded parallel ␤-sheet (S4–S7, in the order 6547) and
six helices (H4–H9). Helix H10, which is a part of the large
domain, has extensive interactions with the small domain. Res-
idues 93–113, helices H5 and H6, and the adjoining loops are in
slightly different conformations in the two protomers. This
region is involved in crystal packing and its similar conforma-
tion in both the protomers would lead to steric clashes. Resi-
dues 279–284 of chain A and 279–283 of chain B were not
modeled due to absence of well defined electron density. These
residues are part of the solvent-exposed loop connecting helix
H12 and H13.
Structuralcomparisonsoftheprotomerwithotherstructuresin
the PDB using DALI server (29) revealed that EcDAPAL is similar
to fold type II PLP-dependent enzymes. The structures most
similar are D-serine deaminase (DSD; PDB code 3R0X) (33),
serine racemase (SR; PDB code 3L6B) (34), L-serine dehydratase
(LSD; PDB code 1PWH) (35), biodegradative threonine deami-
nase (PDB code 2GN1) (36), and biosynthetic threonine deami-
nase (PDB code 1TDJ) (37), with r.m.s.d. in ranges of 2.1–3.0 Å.
Structural comparisons indicated that except for strand S1 of the
large domain and the two helices H5 and H6 of the small domain,
other secondary structural elements are conserved in these
homologs. Apart from EcDAPAL, similar insertions are observed
RESULTS AND DISCUSSION
Subunit Structure from the Tetragonal Crystal Form—The
structure of EcDAPAL has been determined to reasonable
Rwork and Rfree values (Table 1). The structures of the two
protomers of tetragonal crystal form are nearly identical as
revealed by the low r.m.s.d. of 0.24 Å obtained after superposi-
tion of corresponding C␣ atoms of the two chains. As in other
fold type II PLP-dependent enzymes, each protomer consists of
two domains as follows: a large and a small domain, with PLP
bound in a cleft between the two domains (Fig. 2A). The large
domain is made up of segments from both the N (residues
2–69) and C (residues 197–398) termini of the polypeptide,
whereas the small domain is constituted by residues 78–187.
Two loops consisting of residues 70–77 and 188–196, respec-
tively, connect the two domains and are shown in red in Fig. 2A.
Each domain has a ␤-sheet core surrounded by helices. The
large domain is made up of a seven-stranded (S1–S3 and in S. typhimurium DSD (PDB code 3R0X).
JUNE 8, 2012•VOLUME 287•NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 20371

Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 2. Polypeptide fold of EcDAPAL showing secondary structure elements. A, schematic diagram illustrating helices as cylinders and ␤-strands as
ribbons. PLP linked to Lys77 is shown in ball and stick representation. All secondary structure elements are labeled. B, EcDAPAL dimer. The two protomers are
labeled Chain A and Chain B and are colored pink and blue, respectively. C, magnified view of the secondary structure elements involved in interface formation.
The segments of helices and loops involved in the interactions at the dimeric interface are brightly colored.
Interfaces and Biological Unit—EcDAPAL has been shown to different. Two types of quaternary arrangements have been
be a stable dimer in solution (14, 16). The solvent-accessible commonly observed in fold type II enzymes. In interfaces that
surface area of an isolated protomer is about 14,973 Å2. Analy- resemble O-acetylserine sulfhydrylase (38), both small and
sis of all the intermolecular interfaces from the tetragonal crys- large domains are involved in interface formation, and the bur-
tal structure revealed that the largest interface is formed by the ied surface areas range from 1,500–4,000 Å2 in different
two protomers of the asymmetric unit and corresponds to a enzymes. In serine racemase-like interfaces (34), only large
buried surface area of 1,055 Å2 per protomer. This represents domains are involved in the interface, and the buried surface
7.7% of the accessible surface of a protomer. This interface is areas are in the range of 800–1,200 Å2. The dimeric interface of
likely to correspond to the physiological dimer as other inter- EcDAPAL is also formed by interaction of only the large
faces found in the crystal structure have buried areas lower than domains, although the relative arrangement of the two protom-
695 Å2 per monomer. Thus, the two protomers as shown in Fig. ers is different from that of SR. Analysis using the PISA server
2B represent a biological dimer. The interface is formed by the did not lead to the identification of any other enzyme with a
large domains of two protomers related by noncrystallographic similar interface. Hence, EcDAPAL is a dimeric protein with a
2-fold symmetry about an axis inclined to the crystal b by ϳ10°. biological interface that is novel and distinct from interfaces
It involves interactions of residues from four helices and three observed earlier in fold type II PLP-dependent enzymes.
loops of each subunit (Fig. 2C). Residues that are involved in
Active Site—The active site of holo-EcDAPAL is constituted
between the two domains. As established earlier (14), Lys77
intersubunit interactions belong to the N-terminal loop (Ser2 by residues from a single polypeptide chain and occurs in a cleft
and Phe4), H2 (Leu53, Leu56, and Phe57), H14 (Leu319, Arg322
,
Val323, and Gly325), the loop between helix H14 and H15 anchors the co-factor PLP as a Schiff base through its ⑀-amino
(Asn326, Pro327, Tyr328, Arg333, and Ile335), H15 (Tyr352), H17 group. Lys77 is at the N-terminal end of a helix (helix 4, residues
(Tyr385, Arg386, Glu387, and Val389), and the C-terminal loop 78–93) and is also the last residue of the loop (residues 70–77)
(Trp390, Glu391, Gly392, Ala395, and Val396). The interface is sta- that connects the two domains. Apart from this interaction,
bilized by both polar and hydrophobic interactions. Out of the PLP is stabilized by several other noncovalent interactions.
48 residues of the interface, 20 (10 from each subunit) are polar. Oxygen atoms of the phosphate moiety of PLP are hydrogen
20 pairs of hydrogen-bonded residues were found in the inter- bonded to two water molecules, a Tris ion and to the main chain
face (Ser2–Glu387, Ser2–Glu391, Arg322–Trp390, Asn326–Tyr385
Arg333–Asn326, Arg333–Pro327, Arg386–Asn326, Arg386–Pro327
,
,
nitrogen atoms of five residues (Gly232, Val233, Gly234, Ala235
and Met336) occurring in a loop (Fig. 3A). A similar glycine-rich
,
Tyr390–Asn326, Val396–Glu391, and their dyad symmetry loop anchors the phosphate group of PLP in other PLP-depen-
mates).
dent enzymes (39). O3 of PLP is at hydrogen bonding distance
Even though the EcDAPAL protomer shares overall struc- from the side chain nitrogen of Asn122, and N1 is hydrogen
tural similarity with other fold type II PLP-dependent enzymes, bonded to the hydroxyl group of Thr376 (Fig. 3B). The pyridine
the quaternary arrangement of the two subunits of the dimer is ring of PLP is nearly orthogonal to the phenyl ring of Phe76
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Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 3. Active site of EcDAPAL. A and B illustrate lysine-linked PLP at the active site in two different orientations highlighting the interactions of the
phosphategroup(A)andpyridinering(B). Electrondensity(2Fo ϪFc contouredat1.5␴)forthePLP-LysSchiffbasecomplexisshown. C, stereodiagramshowing
the Tris ion bound at the active site of EcDAPAL. Electron density (2Fo Ϫ Fc contoured at 1.5␴) corresponding to the Tris ion is also shown.
leading to a CH-␲ interaction. In most of the fold type II nearly tetragonal geometry to the phosphate group of PLP and
enzymes, pyridine N1 of PLP is hydrogen bonded to a Ser or the main chain oxygen atoms of Gly288 and Ala290. Hydroxyl
Thr. The residue hydrogen bonded to N1 is an Asp or Glu in groups of Tris have hydrogen bonding interactions with car-
fold type I, which enhances the electron withdrawing capacity boxyl groups of Asp120 and Asp189, hydroxyl group of Tyr168
,
of PLP. In contrast, an Arg is hydrogen bonded to N1 in fold main chain nitrogen of Ala290, and three water molecules.
type III enzymes leading to lower electron withdrawing poten- EcDAPAL is less active in Tris buffer compared with phosphate
tial of PLP. These interactions are significant for the functional buffer (data not shown). Similar inhibition in Tris buffer has
ϩ
ϩ
differences between PLP-dependent enzymes of different folds. been reported for DSD (40). NH4 and K prevented inhibition
ϩ
Thus, in EcDAPAL, instead of a carbanion intermediate, a neu- of DSD by Tris in a concentration-dependent manner. Na
tral aminoacrylate is likely to be stable.
ions also prevented the inhibition, although less effectively.
EcDAPAL substrate-binding site is lined by PLP and residues EcDAPAL structure suggests that the observed inhibition is due
from loops 119–123, 188–190, 232–236, 287–291, and 166– to the binding of Tris at the active site and might represent a
169. Lys77, Asp120, Asp189, and Tyr168 are close to the substrate- property shared by several other PLP-dependent enzymes.
binding site and could be important for the catalytic function of
A novel disulfide bond linking Cys265 and Cys291 was
the enzyme. Electron density suitable for a Tris ion was observed close to the substrate access channel in holo-EcDA-
observed at a site close to the phosphate group of PLP in the PAL (Fig. 4A). The homologous enzymes DSD (33), SR (34), and
holo-EcDAPAL structure. The Tris ion fitted to this density LSD (35) bind a metal ion (Na , K , Mg2ϩ, or Mn2ϩ) close to
could be refined with full occupancy and reasonable B-factors. this site with the residue corresponding to Cys265 involved in
It has extensive interactions with the surrounding groups metal coordination. In holo-EcDAPAL, residual density at a
(Fig. 3C). The amino group of Tris is hydrogen bonded with position corresponding to the metal-binding site was observed
ϩ
ϩ
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Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 4. Disulfide bond and the structural differences near the substrate channel between the holo- and apo-EcDAPAL. A, disulfide bond (Cys265
–
Cys291) with respect to the metal ion-binding site found in other fold type II PLP-dependent enzymes. The water molecule bound in EcDAPAL at the site
corresponding to the metal-binding site of other PLP-dependent enzymes is shown as a black ball. Polypeptide chain spanning Cys265–Cys291 is shown in a
schematic representation. Residues 277–283 are disordered and represented by light gray dots. B, schematic diagram showing the substrate channel in
holo-EcDAPAL as a mesh. PLP, Tris, and disulfide-bonded cysteine residues are shown as sticks. Segments that are disordered in apo-EcDAPAL are represented
in dark gray. C, sphere representation of holo-EcDAPAL protomer highlighting the region (in dark) that gets ordered on PLP binding. PLP is shown in a ball and
stick representation. D, superposition of holo- (dark gray) and apo (light gray)-EcDAPAL showing residues that are in different conformation. Part of the region
ordered only in holo-form is shown as a schematic.
in one of the two subunits of the crystallographic asymmetric substrates to the active site. Disulfide bond might limit the dis-
unit. In the SeMet EcDAPAL, both the subunits had density at order in the 261–295 segment. However, further mutational
this site. The density was interpreted as a water molecule based analysis is required to unravel the role of this unique disulfide in
on the distance of this site to the nearest polar groups (Fig. 4A). the function of EcDAPAL.
This suggests that unlike homologous enzymes, there is no
Structure of the Monoclinic Crystal Form—The four protom-
metal ion bound in the vicinity of the EcDAPAL active site. ers (referred to as A, B, C, and D) in the asymmetric unit of the
Instead, a novel disulfide linkage is observed. The metal ion in monoclinic form constitute two dimers similar to the dimeric
these enzymes is likely to stabilize structure of the loop corre- structure observed in the tetragonal form. Superposition of cor-
sponding to residues 261–295 of EcDAPAL. In EcDAPAL, the responding C␣ atoms of different pairs of protomeric units of
disulfide linkage may be important for maintaining the confor- tetragonal and monoclinic forms resulted in r.m.s.d. of 0.4–
mation of the substrate access channel. Residues 279–283, 0.72 Å, showing that the overall structure of all six protomers is
which occur in the solvent-exposed region of the active site similar. The regions with relatively larger differences between
access channel (Fig. 4B), are disordered indicating that the loop the tetragonal and monoclinic forms are 94–110 (helix H5 and
is highly flexible. These residues may regulate the access of H6), 262–293 (helix H12 and subsequent loop), and 164–184
20374 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure and Function of Diaminopropionate Ammonia-lyase
(helix H9) residues. Well defined electron density was absent brought about by loss of PLP favor monoclinic packing
for some of these regions in the monoclinic form, which include arrangement.
residues 109 and 263–293 in chain A; 102–110 and 262–293 in
Structures Obtained after Soaking with DL-DAP—Structure
chain B; 111 and 262–293 in chain C; and 262–291 in chain D. determined from crystals soaked with DL-DAP at pH 8.3 was
Also, electron density for an intact PLP was not observed in any similar to that of the holo-EcDAPAL except at the active site.
of the four protomers. Thus, the tetragonal and monoclinic Superposition of the corresponding C␣ atoms of the two struc-
forms correspond to the holo- and apo-forms of EcDAPAL, tures resulted in an r.m.s.d. of 0.74 Å. The electron density
respectively. However, both the forms resemble the closed form observed at the active site did not correspond to DAP but to
of the fold type II PLP-dependent enzymes. This is in contrast PLP-linked aminoacrylate, a reaction intermediate expected to
to DSD (33) and SR (34) in which the closed form is observed be formed during the catalytic cycle (Fig. 5A). Orientation of
only upon substrate binding.
the pyridine ring of PLP in this structure is related to that of the
The electron density observed at the active site of protomers internal aldimine form by a rotation of 32° (Fig. 5B). In spite of
in the apo-form could be fitted with a phosphate ion and two this rotation, the CH-␲ interaction with Phe76 is maintained.
water molecules. These are bound close to the positions corre- Lys77 side chain is free in this structure, and its ⑀-amino group is
sponding to the PLP phosphate and substrate carboxyl-binding hydrogen bonded to the hydroxyl group of Tyr206, side chain
sites, respectively, of the holo-form. Unlike in the holo-form, carboxyl of Asp189, and phosphate group of PLP. There is no
density corresponding to a Tris ion is absent. As mentioned density for Tyr168 side chain in the A subunit. Apart from these,
above, a stretch of 32 residues (262–293 residues, which include all other active site residues have conformations similar to
the disulfide-bonded cysteines) is disordered in the apo-form, those of the holo-EcDAPAL structure. The carboxyl group of
leaving the active site partially unstructured and exposed to the aminoacrylate makes hydrogen bonding interactions with the
solvent. In the holo-form, this region is mostly ordered and side chain hydroxyl of Thr119 and the main chain N of Asp120
occurs as a helix (H12, residues 265–272) followed by a loop, and His123. Even though the overall structure of the complex is
and only five residues remain disordered (Fig. 4, B and C). These similar to that of holo-EcDAPAL, well defined density was not
results indicate that the structure undergoes disorder to order observed for the region consisting of residues 103–112 and
transition upon binding PLP. The corresponding loop in LSD 104–110, in chains A and B, respectively. This region corre-
has also been shown to assume different conformations in the sponds to helix H6 (residues 102–107) and the subsequent loop.
holo- and apo-forms (35). Residues 31–33 and Asp120 are near
EcDAPAL is the first example of a single substrate fold type II
the active site of the enzyme and are in different conformations PLP-dependent enzyme in which an aminoacrylate has been
in apo- and holo-forms. Residues 31–33 are part of the loop that observed at the active site. PLPꢁaminoacrylate complex has
interacts with the PLP phosphate in the holo structure, and the been observed in five other PLP-dependent enzyme structures
side chain of Asp120 is hydrogen bonded to the main chain N of determined earlier (41–46). Occurrence of aminoacrylate at
Gly288, which is disordered in the apo structure. Binding of PLP the active site of EcDAPAL could be due to the reduced rate of
leads to considerable structural changes leading to shielding of the catalysis in the crystallization condition (see under “Activity
the active site from the bulk solvent and leaving an access chan- Assay and Spectral Studies”). In fold type II enzymes with
nel for the exchange of substrates and products (Fig. 4B).
bound aminoacrylate, the residues corresponding to Lys77 and
Side chains of some other residues away from the active site Thr119 of EcDAPAL are conserved. Except for these similarities,
are also in different orientations in the apo and holo structures. the active site of EcDAPAL is considerably different from those
These include Trp299, Arg303, Phe15, and Lys14 (Fig. 4D). These of other enzymes with bound aminoacrylate.
changes appear to be triggered by the order-disorder transition
Structures obtained for crystals soaked with DL-DAP at pH
between the holo- and apo-forms. Trp299 of the apo structure 6.5 did not reveal additional density at the active site corre-
occupies the same position as that of Ile266, which is ordered sponding to DAP or aminoacrylate, unlike the crystals soaked
only in the holo-form. The changes are largest where the seg- with DL-DAP at pH 8.3. The structure is similar to that of holo-
ment 262–293 is ordered upon binding of PLP and become less EcDAPAL. PLP is linked to the enzyme in the internal aldimine
pronounced toward the surface involved in crystal contacts form, and density corresponding to Tris ion is present. Bio-
near Lys14 (Fig. 4D). Interestingly, the electron density for these chemical studies have indicated that the enzyme degrades DAP
residues is good in the apo-form, and the associated B-factors much more slowly at pH 6.5 than at pH 8.3. Absence of density
are also comparable with the average B-factor for all atoms. In corresponding to the ligand at the active site indicates that low-
the holo-form, these residues have relatively larger B-factors, ering of pH not only affects catalysis but also reduces the affinity
and Lys14 is partially disordered in one of the two protomers of of the enzyme for the substrate. Good electron density corre-
the asymmetric unit.
sponding to Tris ion in both the structures (for crystals soaked
It was found that the holo-dimer is incompatible with the with DL-DAP at pH 8.3 and 6.5) indicates that binding of Tris is
monoclinic crystal packing due to steric clashes involving resi- not affected by pH.
dues 267–278 (in chains C and D) and Lys14 (in chains A, C, and
Structures Obtained after Soaking with D-Ser and L-Ser—
D). Only the structure with disordered segments resulting from Structures of EcDAPAL were also obtained after soaking the
loss of PLP appears to be compatible with the packing arrange- crystals in the presence of D- and L-Ser. Both are substrates for
ment of the monoclinic form. Also, the appearance of only tet- EcDAPAL, although the reaction rates are much lower when
ragonal crystals in the presence of excess PLP during crystalli- compared to that with DAP. In these structures, PLP is bound
zation provides further evidence that structural changes to the enzyme in the internal aldimine form as in the holo-
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Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 5. Stereo diagrams showing active site of EcDAPALꢁligand complexes. A, modeled aminoacrylate with the corresponding electron density (2Fo Ϫ
Fc contoured at 1␴) at the active site in EcDAPAL crystals soaked with DAP. PLPꢁaminoacrylate complex is labeled as PLP-AA. Hydrogen bonds are shown as
dashed lines. B, superposition of the active sites of EcDAPAL (dark gray) and EcDAPAL aminoacrylate complex (light gray). Distances of active site residues from
the C␣ of aminoacrylate are shown (light gray dashes). C, D-Ser with corresponding electron density (2Fo Ϫ Fc contoured at 1␴) at the active site of crystals
soaked with D-Ser. Tris ion bound in the EcDAPAL structure (not soaked with ligands) is shown in a line representation, to illustrate the overlap between the Tris
and the substrate-binding site.
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Structure and Function of Diaminopropionate Ammonia-lyase
TABLE 2
Kinetic parameters of EcDAPAL and its mutants with D- and L-DAP
D-DAP
L-DAP
Km
mM
kcat
k
cat/Km
Km
mM
kcat
kcat/Km
Ϫ1
Ϫ1
Ϫ1
Ϫ1
min
minϪ1mM
min
minϪ1mM
EcDAPAL
D120N
0.05 Ϯ 0.002
2,675 Ϯ 15
ND
5.35 ϫ 104
0.03 Ϯ 0.002
0.57 Ϯ 0.05
11.5 Ϯ 4.4
1,476 Ϯ 76
183 Ϯ 5
11 Ϯ 2
4.92 ϫ 104
3.21 ϫ 102
0.96
NDa
ND
D189N
12.9 Ϯ 2.3
21 Ϯ 4
1.6
a ND means not determined. Activity of D120N EcDAPAL was not detectable with D-DAP as substrate.
EcDAPAL structure. r.m.s.d.s of corresponding C␣ atoms upon (supplemental Fig. S2B). This suggests that under the crystalli-
superposition of D- and L-Ser-soaked structures with the native zation condition, the reaction rate is greatly reduced.
EcDAPAL structure were 0.35 and 0.32 Å, respectively.
Residues that are in close proximity to C␣ of the bound sub-
Structure from crystals soaked with D-Ser had additional strate were mutated by site-directed mutagenesis. The residues
electron density at the substrate-binding site. A molecule of Asp120 and Asp189 were individually mutated to Asn, and Lys77
D-Ser could be fitted into the density with its side chain partially was mutated to both His and Arg. The mutant enzymes were
occupying the Tris-binding site (Fig. 5C). Density correspond- overexpressed and purified by procedures similar to that used
ing to a Tris ion was absent in this structure. The substrate for the wild type enzyme. The purified proteins were checked
carboxyl interacts with the residues of the conserved carboxyl- for the presence of PLP by estimating the absorbance at ϳ412
binding loop. In the external aldimine form, the amino group of nm. No detectable absorbance at 412 nm was observed for the
D-Ser is expected to form a Schiff base with the PLP. The amino Lys mutants, indicating that these did not bind PLP. This illus-
nitrogen of D-Ser is at the distance of ϳ3.5 Å from the C4Ј atom trates the critical role of Lys77 in anchoring PLP at the active
of PLP and is hydrogen bonded to the side chain carboxyl of site. Both the Asp mutants had absorbance maxima at 412 nm
Asp189 and phosphate group of PLP. Asp189 or phosphate group with A412:A280 ratios of 0.08 for D120N and 0.11 for D189N,
of PLP might be important for activation (deprotonation) of the comparable with that of the native holoenzyme (0.12). This
substrate amino group for formation of Schiff base linkage with indicates that these mutations did not interfere with PLP bind-
PLP. The hydroxyl group of D-Ser is at hydrogen bonding dis- ing. Activities of all the mutants were examined using D- and
tance to the main chain oxygen atoms of Gly288 and Ala290. In L-DAP as substrates. Lys mutants K77H and K77R were com-
the structure obtained for the L-Ser-soaked crystals, no ade- pletely inactive toward both the substrates, as expected by their
quate density that could correspond to L-Ser was observed at inability to bind PLP. Kinetic parameters obtained for mutants
the substrate-binding site, suggesting that the enzyme has a D120N and D189N are compared with those of wild type
lower affinity for L-Ser. A Tris ion could be fitted in a confor- EcDAPAL in Table 2. Although D120N EcDAPAL had measur-
mation similar to that of the native structure. In accordance able activity with L-DAP, the catalytic efficiency (kcat/Km) was
with these observations, biochemical assays have shown that reduced by 150-fold as compared with that of native holo-
the Km value for L-Ser is much higher than that for D-Ser (12).
EcDAPAL. No activity could be detected with D-DAP as sub-
Spectral Observations and Activity Measurements—The strate. In contrast, for the wild type enzyme, the catalytic effi-
structure of EcDAPAL obtained from crystals soaked with ciency was nearly the same toward both the stereo isoforms
the substrate (DAP) had a reaction intermediate bound at the (Table 2). Thus, the mutation D120N resulted in a stereospe-
active site. Consistent with this observation, the kcat value of cific enzyme that is active (although lower) only toward the
the enzyme was 360 times lower for DL-DAP in the crystalliza- L-form of the substrate. D189N mutant showed only marginal
tion solution (kcat ϭ ϳ5.6 minϪ1) as compared with that in activity with both the isomers. The catalytic efficiency was 4
phosphate buffer (kcat ϭ ϳ2,038 minϪ1). The enzyme showed orders of magnitude lower with both D- and L-DAP as
no activity with D- or L-Ser in the crystallization solution in substrates.
conformity with the observations on the structures of D-Ser and
To further probe into the effect of mutations on the activity
of the enzyme, the visible absorbance spectra of the wild type
L-Ser EcDAPAL complexes.
Spectral properties of EcDAPAL have earlier been character- and mutant enzymes were recorded as a function of time upon
ized in phosphate buffer (14). Similar experiments were carried addition of substrates. Fig. 6, A and B, corresponds to the spec-
out in the crystallization solution. In phosphate buffer at pH tra of holo-EcDAPAL incubated with D- and L-DAP, respec-
7.5, enzyme showed a characteristic absorbance maximum at tively. As observed earlier, there was a red shift in the absorb-
412 nm, corresponding to the holo-form with PLP bound as an ance maximum from 412 to 422 nm immediately after the
internal aldimine (supplemental Fig. S2A). Upon addition of 10 addition of both D- and L-DAP (Fig. 6, A and B, red line). With
mM DL-DAP, a red shift to 422 nm and a peak at 317 nm were increase in time, this peak reverted back to 412 nm with con-
observed within 15 s. The absorbance maximum reverted to comitant increase in the absorbance at 317 nm (because of the
412 nm and the absorbance at 317 nm stabilized within 10 min product pyruvate). Addition of D-DAP to D120N also resulted
(supplemental Fig. S2A). In contrast, upon addition of 10 mM in a shift of absorbance maximum from 412 to 422 nm, indicat-
DAP in the crystallization condition, no change in the absorb- ing the formation of ketoenamine form of PLP external aldi-
ance at 412 nm was observed, and the peak at 317 nm corre- mine (47). However, no further changes in the spectrum were
sponding to pyruvate increased with time at a much lower rate observed with change in time (Fig. 6C). The small peak
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Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 6. Absorbance spectra of EcDAPAL and mutants upon addition of substrates. A, EcDAPAL ϩ D-DAP. B, EcDAPAL ϩ L-DAP. C, D120N EcDAPAL ϩ
D-DAP. D, D120N EcDAPAL ϩ L-DAP. E, D189N EcDAPAL ϩ D-DAP. F, D189N EcDAPAL ϩ L-DAP. Black line corresponds to the spectrum before the addition of the
substrate. Red, green, blue, and purple lines correspond to the spectra recorded at 15 s and 1, 4, and 10 min, after the addition of the substrate.
observed at 340 nm may correspond to enolimine tautomer of tion is initiated by abstraction of C␣ proton by a general base
PLP (47). The peak at 422 nm was higher than the peak at 412 catalytic group. The ability to recognize both stereo forms of
nm in contrast to that of the wild type enzyme spectrum. Addi- the substrate has been accounted for by either a single base or a
tion of L-DAP to the D120N also resulted in a similar red shift dual base mechanism in other enzymes (48). The single base
and increase in absorbance at 422 nm, which reverted to lower mechanism requires the two isoforms of the substrate to bind in
wavelengths very slowly (Fig. 6D). As with the wild type different orientations at the active site such that the same group
enzyme, increase in absorbance at 317 nm due to the formation on the protein can carry out deprotonation. In the dual base
of pyruvate could be observed. The spectral changes observed mechanism, the two forms of the substrate are held at the active
when D- or L-DAP was added to the D189N mutant were similar site by the same set of interactions, but the enzyme must have
(Fig. 6, E and F). There was a red shift of the peak to 422 nm. The two suitably positioned bases for proton abstraction. Examina-
shifted peak had lower intensity and the peak height reduced tion of the substrate binding pocket of EcDAPAL (Fig. 3) and
marginally with time. The slight increase in absorbance in the the mode of binding of aminoacrylate (Fig. 5, A and B) and D-Ser
317–340-nm region could be a combined effect of product for- (Fig. 5C) suggests that both D- and L-forms of the substrate are
mation and enolimine tautomer of PLP. Taken together, these likely to be held by same set of interactions by the carboxyl-
results suggest that Asp120 might be involved in a step beyond binding loop of the enzyme. In the external aldimine, the ␣-
the formation of external aldimine, probably in the stereo-spe- amino group of the substrate is covalently bonded to the C4Ј
cific proton abstraction from C␣ of D-DAP.
atom of PLP. Therefore, the proton to be abstracted will point
Substrate Binding and Plausible Catalytic Mechanism—It in nearly opposite directions for the two enantiomeric forms of
was shown earlier that EcDAPAL has a narrow reaction and the substrate. The side chains of four residues (Asp120, Asp189
,
substrate specificity (14–16). However, the enzyme can act on Tyr168, and Lys77) are close to the bound substrate and could
both the D- and L-forms of the substrate. In PLP-dependent function as bases for proton abstraction. Although Lys77 is
deaminases, after the formation of external aldimine, the reac- covalently linked to PLP in the internal aldimine form, it is free
20378 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure and Function of Diaminopropionate Ammonia-lyase
FIGURE 7. Catalytic mechanism proposed for EcDAPAL.
to abstract C␣ proton from the PLP substrate external al- sp3-hybridized C␣ atoms of D- and L-DAP are directed toward
dimine. Side chain functional groups of Asp120, Asp189, Lys77, Asp120 and Lys77, respectively. Abstraction of the C␣ proton
and Tyr168 are at distances of 3.4, 4.1, 4.4, and 5.4 Å, respec- would lead to the formation of a transient carbanion interme-
tively, from the C␣ of aminoacrylate. The distance and orienta- diate. As the carbanion intermediate is unlikely to be stable in
tion of Tyr168 is not appropriate for the abstraction of the pro- EcDAPAL, abstraction of C␣ proton may lead to a concomitant
ton. Furthermore, Tyr168 is not conserved in SR and LSD, release of the ␤-amino group as an ammonium ion, and a PLP-
indicating that it is not crucial for catalysis. Asp120 faces the si aminoacrylate intermediate would be formed. Aminoacrylate
side and side chains of Lys77 and Asp189 are on the re side of would be released by the neutral side chain of Lys77 approach-
aminoacrylate. Mutational studies also show that Asp120 is cru- ing the C4Ј atom of PLP and forming a Schiff base linkage. The
cial for activity toward D-DAP. Asp120 corresponds to a threo- released aminoacrylate may then be deaminated to pyruvate by
nine in DSD (33) and serine in SR (34). These residues have also either nonenzymatic hydrolysis (49) or by assistance of other
been proposed to abstract proton from their respective D-iso- cellular proteins (50).
mer of substrates. Activity assays with D189N mutants indi-
Conclusions—The work presented here has revealed key dif-
cated that Asp189 was important for catalysis. However, the loss ferences between EcDAPAL and other fold type II PLP-depen-
of activity was comparable with both isoforms, indicating that dent enzymes. EcDAPAL is a dimeric protein with an interface
Asp189 might be important for binding of the substrate but may that has not been observed earlier in other fold type II PLP-de-
not be responsible for abstraction of the proton from the L-iso- pendent enzymes. EcDAPAL has a novel disulfide bond close to
mer of substrate. The ⑀-amino group of Lys77 can approach the the site at which a metal ion binds in other fold type II PLP-de-
C␣ of the bound substrate by slight changes in the side chain pendent enzymes. The disulfide bond or the metal-binding site
conformation. Corresponding lysine residues in SR (34), LSD could be important for stabilizing the loops lining the substrate
(35), and biodegradative threonine deaminase (36) have been channel. Structures of the PLP-bound and -unbound form of
proposed to abstract the proton from the L-isoform of their the enzyme highlighted important structural differences near
respective substrates. Therefore, in EcDAPAL Asp120 and Lys77 the active site and substrate channel, which might represent the
are potential catalytic residues for the abstraction of C␣ pro- transition that the enzyme undergoes upon binding of PLP.
tons from the D- and L-isoforms of substrate, respectively.
Unlike other homologs (which undergo transition from open to
Based on the structures of EcDAPAL and its comparison closed form on substrate binding), all structures of EcDAPAL
with homologous enzymes, a plausible catalytic scenario could resemble a closed form. Examination of catalytic properties of
be envisaged (Fig. 7). The negatively charged active site of EcDAPAL and active site geometry of the holo-form suggests
EcDAPAL is suitable for the binding of the positively charged that the Tris ion is likely to be an inhibitor of PLP-dependent
substrate DAP. EcDAPALꢁD-Ser complex structure suggests enzymes. Soaking EcDAPAL with D-Ser revealed a molecule of
that DAP may be initially held by interactions of its carboxylate D-Ser bound at the active site without forming a Schiff base
group with the conserved loop (119TAGNH123). The amino linkage to PLP. This represents the initial mode of binding of
group of DAP is then deprotonated by PLP phosphate or substrate to the active site before being covalently attached to
Asp189. The neutral amino group is suitable for nucleophilic PLP. Structure determined from EcDAPAL crystals soaked
attack on the C4Ј atom of PLP leading to the release of Lys77. with DL-DAP provides direct evidence for the formation of
Concomitantly, the pyridine ring of PLP is tilted by 32° toward aminoacrylate intermediate in the reaction. The work has high-
the solvent side forming an external aldimine. This brings the lighted the importance of Lys77, Asp120, and Asp189 in the func-
substrate to the orientation represented by EcDAPALꢁ tion of the enzyme. Asp120 and Lys77 are likely to be the residues
aminoacrylate complex. In this position, the ␣-protons on the responsible for proton abstraction from C␣ of D- and L-DAP,
JUNE 8, 2012•VOLUME 287•NUMBER 24
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Structure and Function of Diaminopropionate Ammonia-lyase
Methods Enzymol. 276, 307–326
respectively. Further structural studies on mutant enzymes and
their complexes will provide deeper insights into this unique
PLP-dependent enzyme.
20. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols,
N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., Mc-
Coy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C.,
Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX. A
comprehensive Python-based system for macromolecular structure solu-
tion. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221
Acknowledgments—We thank the staff at the X-ray Laboratory at
Molecular Biophysics Unit and the beamline scientists at ESRF and
SPRING-8 for their cooperation during data collection. We acknowl-
edge J. Rajaganapathi for help during the initial stages of the work.
21. Grosse-Kunstleve, R. W., and Adams, P. D. (2003) Substructure search
procedures for macromolecular structures. Acta Crystallogr. D Biol. Crys-
tallogr. 59, 1966–1973
22. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Sto-
roni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl.
Crystallogr. 40, 658–674
REFERENCES
1. Gmelin, R., Strauss, G., and Hasenmaier, G. (1959) New amino acids from
Mimosaceae. Hoppe-Seyler’s Z. Physiol. Chem. 314, 28–32
2. Wada, S., and Toyota, T. (1965) Isolation of D(Ϫ)-2,3-diaminopropionic
acid from digestive fluid of silkworm larvae, Bombyx mori. Biochem. Bio-
phys. Res. Commun. 19, 482–486
23. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of
macromolecular structures by the maximum likelihood method. Acta
Crystallogr. D Biol. Crystallogr. 53, 240–255
24. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and
development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501
25. Collaborative Computational Project No. 4 (1994) The CCP4 suite. Pro-
grams for protein crystallography. Acta Crystallogr. D Biol. Crystallogr.
50, 760–763
3. Haskell, T. H., Fusari, S. A., Frohardt, R. P., and Bartz, Q. R. (1952) The
chemistry of viomycin 1. J. Am. Chem. Soc. 74, 599–602
4. Wang, M., and Gould, S. J. (1993) Biosynthesis of capreomycin. 2. Incor-
poration of L-serine, L-alanine, and L-2,3-diaminopropionic acid. J. Org.
Chem. 58, 5176–5180
26. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M.
(1993) PROCHECK. A program to check the stereochemical quality of
protein structures. J. Appl. Crystallogr. 26, 283–291
5. Zhao, C., Song, C., Luo, Y., Yu, Z., and Sun, M. (2008) L-2,3-diaminopro-
pionate. One of the building blocks for the biosynthesis of Zwittermicin A
in Bacillus thuringiensis subsp. kurstaki strain YBT-1520. FEBS Lett. 582,
3125–3131
27. Cohen, G. E. (1997) ALIGN. A program to superimpose protein coordi-
nates, accounting for insertions and deletions. J. Appl. Crystallogr. 30,
1160–1161
6. Cheema, P. S., Malathi, K., Padmanaban, G., and Sarma, P. S. (1969) The
neurotoxicity of ␤-N-oxalyl-L-␣,␤-diaminopropionic acid, the neurotoxin
from the pulse Lathyrus sativus. Biochem. J. 112, 29–33
7. Spencer, P. S., Roy, D. N., Ludolph, A., Hugon, J., Dwivedi, M. P., and
Schaumburg, H. H. (1986) Lathyrism. Evidence for role of the neuroexcit-
atory amino acid BOAA. Lancet 2, 1066–1067
28. Krissinel, E., and Henrick, K. (2004) Secondary-structure matching (SSM),
a new tool for fast protein structure alignment in three dimensions. Acta
Crystallogr. D Biol. Crystallogr. 60, 2256–2268
29. Holm, L., and Rosenström, P. (2010) Dali server. Conservation mapping in
three dimensions. Nucleic Acids Res. 38, W545–W549
30. Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assem-
blies from crystalline state. J. Mol. Biol. 372, 774–797
8. Malathi, K., Padmanaban, G., and Sarma, P. S. (1970) Biosynthesis of ␤-N-
oxalyl-L-␣,␤-diaminopropionic acid, the Lathyrus sativus neurotoxin.
Phytochemistry 9, 1603–1610
31. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano
Scientific LLC, San Carlos, CA
9. Corrigan, J. J., and Srinivasan, N. G. (1966) The occurrence of certain
D-amino acids in insects. Biochemistry 5, 1185–1190
32. Shenoy, A. R., and Visweswariah, S. S. (2003) Site-directed mutagenesis
using a single mutagenic oligonucleotide and DpnI digestion of template
DNA. Anal. Biochem. 319, 335–336
10. Grishin, N. V., Phillips, M. A., and Goldsmith, E. J. (1995) Modeling of the
spatial structure of eukaryotic ornithine decarboxylases. Protein Sci. 4,
1291–1304
33. Bharath, S. R., Bisht, S., Savithri, H. S., and Murthy, M. R. (2011) Crystal
structures of open and closed forms of D-serine deaminase from Salmo-
nella typhimurium. Implications on substrate specificity and catalysis.
FEBS J. 278, 2879–2891
11. Jansonius, J. N. (1998) Structure, evolution, and action of vitamin B6-de-
pendent enzymes. Curr. Opin. Struct. Biol. 8, 759–769
12. Vijayalakshmi, K. R., Rao, D. R., and Rao, M. R. (1975) Studies on a 2,3-
diaminopropionate ammonia-lyase from a Pseudomonad. Hoppe-Seyler’s
Z. Physiol. Chem. 356, 193–201
34. Smith, M. A., Mack, V., Ebneth, A., Moraes, I., Felicetti, B., Wood, M.,
Schonfeld, D., Mather, O., Cesura, A., and Barker, J. (2010) The structure
of mammalian serine racemase. Evidence for conformational changes
upon inhibitor binding. J. Biol. Chem. 285, 12873–12881
13. Mushahwar, I. K., and Koeppe, R. E. (1973) Rat liver L-diaminopropionate
ammonia-lyase. Identification as cystathionase. J. Biol. Chem. 248,
7407–7411
35. Yamada, T., Komoto, J., Takata, Y., Ogawa, H., Pitot, H. C., and Takusa-
gawa, F. (2003) Crystal structure of serine dehydratase from rat liver. Bio-
chemistry 42, 12854–12865
14. Khan, F., Jala, V. R., Rao, N. A., and Savithri, H. S. (2003) Characterization
of recombinant diaminopropionate ammonia-lyase from Escherichia coli
and Salmonella typhimurium. Biochem. Biophys. Res. Commun. 306,
1083–1088
36. Simanshu, D. K., Savithri, H. S., and Murthy, M. R. (2006) Crystal struc-
tures of Salmonella typhimurium biodegradative threonine deaminase
and its complex with CMP provide structural insights into ligand-induced
oligomerization and enzyme activation. J. Biol. Chem. 281, 39630–39641
37. Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chin-
chilla, D., and Eisenstein, E. (1998) Structure and control of pyridoxal
phosphate-dependent allosteric threonine deaminase. Structure 6,
465–475
15. Nagasawa, T., Tanizawa, K., Satoda, T., and Yamada, H. (1988) Diamino-
propionate ammonia-lyase from Salmonella typhimurium. Purification
and characterization of the crystalline enzyme, and sequence determina-
tion of the pyridoxal 5Ј-phosphate-binding peptide. J. Biol. Chem. 263,
958–964
16. Uo, T., Yoshimura, T., Nishiyama, T., and Esaki, N. (2002) Gene cloning,
purification, and characterization of 2,3-diaminopropionate ammonia- 38. Burkhard, P., Rao, G. S., Hohenester, E., Schnackerz, K. D., Cook, P. F., and
lyase from Escherichia coli. Biosci. Biotechnol. Biochem. 66, 2639–2644
17. Doublié, S. (1997) Preparation of selenomethionyl proteins for phase de-
termination. Methods Enzymol. 276, 523–530
Jansonius, J. N. (1998) Three-dimensional structure of O-acetylserine sul-
fhydrylase from Salmonella typhimurium. J. Mol. Biol. 283, 121–133
39. Denesyuk, A. I., Denessiouk, K. A., Korpela, T., and Johnson, M. S. (2002)
Functional attributes of the phosphate group binding cup of pyridoxal
phosphate-dependent enzymes. J. Mol. Biol. 316, 155–172
18. Rajaram, V., Rajaganapathi, J., Khan, F., Savithri, H. S., and Murthy, M. R.
(2003) Crystallization and preliminary x-ray diffraction studies on recom-
binant diaminopropionate ammonia-lyase from Escherichia coli. Acta 40. Dupourque, D., Newton, W. A., and Snell, E. E. (1966) Purification and
Crystallogr. D Biol. Crystallogr. 59, 1668–1669
properties of D-serine dehydrase from Escherichia coli. J. Biol. Chem. 241,
19. Otwinowski, Z., and Minor, W. (1997) Processing of x-ray diffraction data
1233–1238
collected in oscillation mode. (Macromolecular crystallography. Part A). 41. Shimon, L. J., Rabinkov, A., Shin, I., Miron, T., Mirelman, D., Wilchek,
20380 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 24•JUNE 8, 2012

Structure and Function of Diaminopropionate Ammonia-lyase
M., and Frolow, F. (2007) Two structures of alliinase from Alliium
282, 23473–23481
sativum L.. Apo-form and ternary complex with aminoacrylate reac- 46. Koutmos, M., Kabil, O., Smith, J. L., and Banerjee, R. (2010) Structural
tion intermediate covalently bound to the PLP cofactor. J. Mol. Biol.
366, 611–625
basis for substrate activation and regulation by cystathionine ␤-synthase
(CBS) domains in cystathionine ␤-synthase. Proc. Natl. Acad. Sci. U.S.A.
107, 20958–20963
42. Clausen, T., Kaiser, J. T., Steegborn, C., Huber, R., and Kessler, D. (2000)
Crystal structure of the cystine C-S lyase from Synechocystis. Stabilization 47. Mozzarelli, A., and Bettati, S. (2006) Exploring the pyridoxal 5Ј-phos-
of cysteine persulfide for FeS cluster biosynthesis. Proc. Natl. Acad. Sci.
U.S.A. 97, 3856–3861
phate-dependent enzymes. Chem. Rec. 6, 275–287
48. Watanabe, A., Yoshimura, T., Mikami, B., Hayashi, H., Kagamiyama, H.,
and Esaki, N. (2002) Reaction mechanism of alanine racemase from Ba-
cillus stearothermophilus. X-ray crystallographic studies of the enzyme
bound with N-(5Ј-phosphopyridoxyl)alanine. J. Biol. Chem. 277,
19166–19172
43. Schneider, T. R., Gerhardt, E., Lee, M., Liang, P. H., Anderson, K. S., and
Schlichting, I. (1998) Loop closure and intersubunit communication in
tryptophan synthase. Biochemistry 37, 5394–5406
44. Ngo, H., Kimmich, N., Harris, R., Niks, D., Blumenstein, L., Kulik, V.,
Barends, T. R., Schlichting, I., and Dunn, M. F. (2007) Allosteric regulation 49. Hillebrand, G. G., Dye, J. L., and Suelter, C. H. (1979) Formation of an
of substrate channeling in tryptophan synthase. Modulation of the L-ser-
ine reaction in stage I of the ␤-reaction by ␣-site ligands. Biochemistry 46,
7740–7753
intermediate and its rate of conversion to pyruvate during the tryptopha-
nase-catalyzed degradation of S-o-nitrophenyl-L-cysteine. Biochemistry
18, 1751–1755
45. Schnell, R., Oehlmann, W., Singh, M., and Schneider, G. (2007) Structural 50. Lambrecht, J. A., Flynn, J. M., and Downs, D. M. (2012) Conserved YjgF
insights into catalysis and inhibition of O-acetylserine sulfhydrylase from
Mycobacterium tuberculosis. Crystal structures of the enzyme ␣-amino-
acrylate intermediate and an enzyme-inhibitor complex. J. Biol. Chem.
protein family deaminates reactive enamine/imine intermediates of pyri-
doxal 5Ј-phosphate (PLP)-dependent enzyme reactions. J. Biol. Chem.
287, 3454–3461
JUNE 8, 2012•VOLUME 287•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 20381

Enzymology:
Crystal Structure of Escherichia coli
Diaminopropionate Ammonia-lyase
Reveals Mechanism of Enzyme Activation
and Catalysis
Shveta Bisht, Venkatesan Rajaram,
Sakshibeedu R. Bharath, Josyula Nitya
Kalyani, Farida Khan, Appaji N. Rao,
Handanahal S. Savithri and Mathur R. N.
Murthy
J. Biol. Chem. 2012, 287:20369-20381.
doi: 10.1074/jbc.M112.351809 originally published online April 13, 2012
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