D.V. Urusova et al. / Biochimica et Biophysica Acta 1824 (2012) 422–432
431
Therefore, at present the catalytic mechanism of DSD has to be putative
Acknowledgement
and the reported distances are only tentatively. The catalytic mechanism
is, however, very likely to be very similar to that proposed for rat liver
L-serine dehydratase [20] or serine racemase [19].
We thank Prof. Manfred Christl, Institute of Organic Chemistry,
University of Würzburg, for his help with the construction of Fig. 9.
We acknowledge the use of the following synchrotron facilities:
SRS Daresbury beamline 10.1, EMBL/DESY beamline X31 and MPG/
DESY beamline BW6.
The phosphate group of PLP is located in a pocket coordinated to
main chain amides of conserved glycines (279GlyValGlyGlyGly283).
The substrate D-serine enters the active site from the re face with re-
spect to the pyridine ring of the cofactor [49]. The amino group of the
substrate, D-serine, forms a hydrogen bond with an oxygen of the co-
factor phosphate (Fig. 9, 1). The phosphate becomes protonated and
the amino group changes into a nucleophile, attacking the C4′ of the
internal Schiff base by releasing the neutral Lys118 (Figs. 9, 2). The
O1 of the carboxyl group of D-serine is coordinated to the hydroxyl
group of Ser167 (3.3 and 2.8 Å, respectively) (Fig. 8) and the amide
nitrogen of Leu171 (2.8 Å) via hydrogen bonds. The O2 of the carbox-
yl group of D-serine is hydrogen-bonded to the hydroxyl group of
Ser167 (3.3 Å) and the amide nitrogen of Thr168 (3.4 Å). The hydrox-
yl group of D-serine is hydrogen-bonded to the carboxyl group of
Asp238 (2.7 Å) and to the phosphate group of PLP via a water mole-
cule (Fig. 8). The N1 of PLP is not protonated, the abstraction of the
α-hydrogen from the Cα of D-serine cannot be executed by Lys118,
because the ε-amino group resides in the si face whereas the α-proton
of the substrate protudes to the re face of the D-serine-PLP Schiff base.
It is very likely that the phosphate group of PLP can act as a general
acid to donate a proton to the oxygen OG of D-serine and abstraction
of the hydrogen from Cα by the hydroxyl group of Thr168 occurs in a
concerted fashion (Fig. 9, 4). Water and the PLP–α-aminoacrylate Schiff
base are released (Fig. 9, 4). Then, Lys118 attacks C4′ of the Schiff base of
the PLP–α-aminoacrylate intermediate to form the PLP-Lys118 (inter-
nal) Schiff base (Fig. 9, 5) and releases α-aminoacrylate (Fig. 9, 6)
which in a non-enzymatic hydrolysis is deaminated to pyruvate. The
putative mechanism of the reaction is summarized in Fig. 9. The catalytic
mechanism of DSD is very much like that of LSD [39] the only difference
is that in the case of DSD Thr168 abstracts the proton from the Cα of D--
serine whereas in LSD Lys 41 is responsible for the abstraction. The
hydroxyl group of D-serine is forming a hydrogen bond to a water
molecule (3.3 Å) and the distance from the water molecule to the
oxygens of the cofactor phosphate is 2.6 and 3.2 Å, respectively.
Other examples of the use of the cofactor phosphate group as acid–
base catalyst are L-serine dehydratase [45] and glycogen phosphory
lase [50].
Alanine racemase is an example of a PLP-dependent enzyme
with unique features of its active site. The pyridine nitrogen of the co-
factor is unprotonated because it accepts a hydrogen bond from
Arg219. This structural feature makes it difficult to form a charge-
delocalized quinonoid intermediate as in other PLP-dependent en-
zymes, yet alanine racemase can still effectively lower the α-amino
carbon acidity and enhance the reaction rate of proton transfer reac-
tions [51,52].
In the two structures of D-serine deaminase from S. typhimurium
reported by Bharath and coworkers [5], no significant electron den-
sity was observed for the cofactor PLP, indicating that the enzyme
has a low affinity for the cofactor or even represents the apoenzyme
form, under the crystallization conditions used. Electron density
corresponding to a plausible sodium ion was seen near the active
site of the closed conformation of the deaminase. It is not quite
clear why the authors use sodium and not potassium ions during
crystallization. The reason might be that the enzymatic activity of
the S. typhimurium enzyme with D-serine as substrate is higher in
the presence of Na+ than K+ ions. We used potassium in all steps of
the protein purification of DSD from E. coli [53] and thus think that
the electron densities observed for the monovalent cation binding
sites are occupied by potassium. Future studies including site-directed
mutagenesis of amino acid residues, such as Asp238, Thr168 and
Ser167, will shed additional light on their involvement in the catalytic
mechanism.
References
[1] W. Dowhan Jr., E.E. Snell, D-Serine dehydratase from Escherichia coli. II. Analytical
studies and subunit structure, J. Biol. Chem. 245 (1970) 4618–4628.
[2] W.K. Maas, B.D. Davis, Pantothenate studies. I. Interference by D-serine and L-
aspartic acid with pantothenate synthesis in Escherichia coli, J. Bacteriol. 60
(1950) 733–740.
[3] Y.Z. Huang, E.E. Snell, D-Serine dehydratase from Escherichia coli. IV. Comparative
sequences of pyridoxylpeptides derived from the active site and from the inhibi-
tory site of the enzyme, J. Biol. Chem. 247 (1972) 7358–7364.
[4] N.V. Grishin, M.A. Phillips, E.J. Goldsmith, Modeling of the spatial structure of eu-
karyotic ornithine decarboxylases, Protein Sci. 4 (1995) 1291–1304.
[5] G. Obmolova, A. Tepliakov, E. Harutyunyan, G.G. Wahler, K.D. Schnackerz, Crystal-
lization and preliminary X-ray studies of D-serine dehydratase from Escherichia
coli, J. Mol. Biol. 214 (1990) 641–642.
[6] H. Tanaka, M. Senda, N. Venugopalan, A. Yamamoto, T. Senda, T. Ishida, K. Horiike,
Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney,
J. Biol. Chem. 286 (2011) 27548–27558.
[7] T. Ito, H. Hemmi, K. Kataoka, Y. Mukai, T. Yoshimura, A novel zinc-dependent D-
serine dehydratase from Saccharomyces cerevisiae, Biochem. J. 409 (2008)
399–406.
[8] S.R. Bharath, S. Bisht, H.S. Savithri, M.R.N. Murthy, Crystal structures of open and
closed forms of D-serine deaminase from Salmonella typhimurium — implications
on substrate specificity and catalysis, FEBS J. 278 (2011) 2879–2891.
[9] M. Marceau, E. McFall, S.D. Lewis, J.A. Shafer, D-Serine dehydratase from Escheri-
chia coli. DNA sequences and identification of catalytically inactive glycine to
aspartic acid variants, J. Biol. Chem. 263 (1988) 16926–16933.
[10] E. Schiltz, K.D. Schnackerz, Sequence studies on D-serine dehydratase of Escheri-
chia coli. Primary structure of the tryptic phosphopyridoxyl peptide and of the N-
terminus, Eur. J. Biochem. 71 (1976) 109–116.
[11] A.A. Vagin, A. Teplyakov, MOLREP: an automated program for molecular replace-
ment, J. Appl. Crystallogr. 30 (1997) 1022.
[12] D.T. Gallagher, G.L. Gilliland, G. Xiao, J. Zondlo, K.E. Fisher, D. Chinchilla, E. Eisen-
stein, Structure and control of pyridoxal phosphate dependent allosteric threo-
nine deaminase, Structure 6 (1998) 465–475.
[13] CCP4. Collaborative Computational Project, Number 4, The CCP4 suite: programs
for protein crystallography, Acta Crystallogr. D50 (1994) 760–763.
[14] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular struc-
tures by the maximum-likelihood method, Acta Crystallogr. D Biol. Crystallogr.
53 (1997) 240–255.
[15] K. Cowtan, P. Main, Miscellaneous algorithms for density modification, Acta Crys-
tallogr. D Biol. Crystallogr. 54 (1998) 487–493.
[16] V.S. Lamzin, K.S. Wilson, Automated refinement of protein models, Acta Crystal-
logr. D Biol. Crystallogr. 49 (1993) 129–147.
[17] T.A. Jones, J.Y. Zou, S.W. Cowan, Kjeldgaard, Improved methods for building pro-
tein models in electron density maps and the location of errors in these models,
Acta Crystallogr. A 47 (1991) 110–119.
[18] K.D. Schnackerz, K. Feldmann, W.E. Hull, Phosphorus-31 nuclear magnetic reso-
nance study of D-serine dehydratase: pyridoxal phosphate binding site, Biochem-
istry 18 (1979) 1536–1539.
[19] M. Goto, T. Yamauchi, N. Kamiya, I. Miyahara, T. Yoshimura, H. Mihara, T. Kurihara, K.
Hirotsu, N. Esaki, Crystal structure of a homolog of mammalian serine racemase from
Schizosaccharomyces pombe, J. Biol. Chem. 284 (2009) 25944–25952.
[20] T. Yamada, J. Komoto, Y. Takata, H. Ogawa, H.C. Pitot, F. Takusagawa, Crystal
structure of serine dehydratase from rat liver, Biochemistry 42 (2003)
12854–12865.
[21] K.D. Schnackerz, J.W. Keller, R.S. Phillips, M.D. Toney, Ionization state of pyridoxal
5′-phosphate in D-serine dehydratase, dialkylglycine decarboxylase, and tyrosine
phenol-lyase and the influence of monovalent cations as inferred by 31P NMR
spectroscpopy, Biochim. Biophys. Acta 1764 (2006) 230–238.
[22] A.T. Diekers, D. Niks, I. Schlichting, M.F. Dunn, Tryptophan synthase: structure
and function of the monovalent cation site, Biochemistry 48 (2009)
10997–11010.
[23] A. Hashimoto, T. Nishikawa, T. Hayashi, N. Fujii, K. Harada, T. Oka, K. Takashi, The
presence of free D-serine in rat brain, FEBS Lett. 296 (1992) 33–36.
[24] H. Wolosker, E. Dumin, L. Balan, V.N. Foltyn, D-Amino acids in the brain: D-serine
in neurotransmission and neurodegeneration, FEBS J. 275 (2008) 3514–3526.
[25] E.C. Gustafson, E.R. Stevens, H. Wolosker, R.F. Miller, Endogeneous D-serine con-
tributes to NMDA-receptor-mediated light-evoked responses in the vertebrate
retina, J. Neurophysiol. 98 (2007) 122–130.
[26] K. Hashimoto, T. Fukushima, T. Shimizu, N. Komatsu, H. Watanabe, N. Shinoda, M.
Nakazato, C. Kumakiri, S. Okada, H. Hasegawa, H. Imai, M. Iyo, Decreased serum
levels of D-serine in patients with schizophrenia: evidence in support of the N-
methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia, Arch.
Gen. Psychiatry 60 (2003) 572–576.