Department of Biochemistry, University of Cambridge, Tennis Court Road, The Downing Site, Cambridge CB2 1QW, UK1
Author for correspondence: Cesar A. Arias. Tel: +57 1 633 1512. Fax: +1 917 477 3388. e-mail: caa22{at}cable.net.co
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ABSTRACT |
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Keywords: D-serine, vancomycin resistance, racemases, Enterococcus gallinarum
a Present address: Centro de Investigaciones, Universidad El Bosque, Transversal 9A no. 133-25, Santafé de Bogotá, Colombia.
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INTRODUCTION |
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D-Ser is not a naturally occurring amino acid, although it is produced spontaneously in the mammalian stomach due to low pH values, and it has also been reported to be a growth inhibitor of Escherichia coli in minimal media (McFall & Newman, 1996 ). Its presence has been reported in E. coli strains, where it serves as a readily available source of carbon (Gutnick et al., 1969
): a specific deaminase system converts D-Ser to pyruvate (Dowhan & Snell, 1970
). Alanine racemases are pyridoxal-phosphate-dependent enzymes that catalyse the production of D-Ala from its enantiomer precursor L-Ala (Walsh, 1989
). Two different alanine racemases (anabolic and catabolic) have been characterized in Salmonella typhimurium and E. coli (Wasserman et al., 1983
; Wild et al., 1985
) whereas in other bacteria investigated only one alanine racemase has been detected. The serine racemase activity of the anabolic enzyme from S. typhimurium involved in the synthesis of peptidoglycan was only 15% of the alanine racemase activity (Esaki & Walsh, 1986
). In multicellular organisms pyridoxal-phosphate-dependent serine racemases have been characterized in the silkworm Bombyx mori (Uo et al., 1998
), where the concentration of D-Ser is increased at particular stages of metamorphosis (Corrigan & Srinivasan, 1966
), and in mammalian brain (Wolosker et al., 1999a
), where it functions as a neuromodulator at the glycine site of the N-methyl-D-aspartate receptor (Matsui et al., 1995
). VanT is the only serine racemase identified in bacteria so far. Unlike other alanine and serine racemases described, VanT is a transmembrane protein: at least ten transmembrane helices are predicted to be present in the N-terminal domain (Arias et al., 1999
) and the C-terminal domain has structural homology with the alanine racemase from Bacillus stearothermophilus (Shaw et al., 1997
), including conservation of the most important residues necessary for binding of the pyridoxal phosphate cofactor (Lys371) and for catalytic activity. Molecular modelling demonstrated that VanT could exist as a dimer (Arias et al., 1999
). In this paper we demonstrate that VanT exhibits significant alanine racemase activity and confirm that the C-terminal domain is sufficient for racemase activity.
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METHODS |
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Functional complementation experiments were carried out in E. coli TKL-10 and the same strain containing plasmid constructs expressing vanT (pCA4) and alr (pJW40). Bacteria were grown at 42 °C (non-permissive temperature) and at 30 °C simultaneously in LB agar and broth and incubated for at least 72 h.
DNA manipulations and plasmid construction.
Cloning, digestion with restriction endonucleases, isolation of plasmid DNA, transformation and ligations were carried out by standard methods (Sambrook et al., 1989 ). Plasmids pCA4 and pCA5 have been described previously (Arias et al., 1999
). For construction of plasmid pJW40, the alr gene from B. stearothermophilus was amplified from total DNA with primer A (5'-GATCGATCGATCCATATGAACGACTTTCATCGCGATACG-3'), which includes a NdeI site (underlined), and primer B (5'-GATCGAGGATCCAAGCTTTTAGTGATGGTGATGGTGATGTGCACTGCTTTCCCCGCGGCC-3'), which includes a BamHI site (underlined), a HindIII site (bold) and the sequence encoding six histidines followed by a stop codon (italics). The PCR product was cloned as a NdeIHindIII fragment into a derivative of pUC19 (Norrander et al., 1983
) under the control of the trc promoter from pKK233-2 (Amann & Brosius, 1985
).
Preparation of membrane and cytoplasmic extracts.
E. coli TKL-10, E. coli XL-1 Blue and derivatives were grown in LB medium (25 ml) at 34 °C with aeration, with corresponding antibiotics if needed. When the OD600 had reached 0·8, 0·2 mM IPTG was added and incubation continued for 2·5 h. Bacteria were harvested, washed in 150 mM Bistris propane buffer (pH 7·5), resuspended in 1 ml of the same buffer and sonicated. The broken cell preparation was centrifuged at 100000 g for 60 min, the supernatant (cytoplasmic fraction) was collected and the pellet (membrane fraction) was washed in 150 mM Bistris propane buffer (pH 7·5) before final resuspension in 200 µl of the same buffer.
Purification of the C-terminal domain of VanT.
E. coli M15[pREP4] (Qiagen) containing plasmid pCA5 encoding the His-tagged C-terminal domain of VanT (C-VanT) (Arias et al., 1999 ) was grown at 37 °C in LB broth (300 ml) containing sorbitol (0·5 M) and betaine (2·5 mM) to favour the synthesis of soluble protein (Blackwell & Horgan, 1991
), due to the fact that initial purification experiments indicated the presence of inclusion bodies when the gene was expressed after induction with IPTG. When the OD600 had reached 1·0, 0·05 mM IPTG was added and incubation continued for 40 min. Bacteria were harvested at 4 °C, washed in lysis buffer (50 mM Bistris propane/300 mM NaCl/10 mM imidazole, pH 8·0) resuspended in 5 ml of the same buffer and sonicated. The broken cell preparation was centrifuged at 40000 g for 20 min at 4 °C and the supernatant collected. All the following steps were carried out at 4 °C. The supernatant was applied to a 2 ml nickel-containing agarose column (Agarose Ni-NTA, Qiagen) equilibrated with the same buffer. The column was washed with at least 100 ml 50 mM Bistris propane/300 mM NaCl/30 mM imidazole buffer (pH 8) and His-tagged C-VanT eluted with 50 mM Bistris propane/300 mM NaCl/250 mM imidazole buffer (pH 8·0). Fractions of 0·5 ml were collected and assayed for serine racemase activity (see below).
Electrophoresis.
SDS-PAGE on a 12% polyacrylamide gel was carried out under denaturing conditions using the Laemmli buffer system (Laemmli, 1970 ) to determine the purity and Mr of C-VanT. For calibration, standard proteins in the range of Mr 9000175000 (New England Biolabs) were used. Proteins were stained with 0·1% (w/v) Coomassie blue in 50% (v/v) methanol/10% (v/v) acetic acid for 30 min at 37 °C and destained with 10% (v/v) methanol/10% (v/v) acetic acid at room temperature overnight.
N-terminal sequencing.
The possibility of heterodimer formation between subunits of C-VanT and endogenous alanine racemases of E. coli was investigated by N-terminal sequencing of the major band corresponding to the His-tagged C-VanT (Mr 43087) and a band with an Mr similar to the anabolic or catabolic alanine racemases (Mr 39128 and 38819 respectively) of E. coli. Proteins present in the sample containing the purified His-tagged C-VanT were precipitated with 10% (v/v) trichloroacetic acid (TCA), separated on a 12% SDS-PAGE gel and electrotransferred to a PVDF membrane. The membrane was stained with 0·1% (w/v) Coomassie blue in 50% (v/v) methanol/1% (v/v) acetic acid, destained with 50% methanol, washed in distilled water and dried. The upper and lower portions of the His-tagged C-VanT and of a band with slightly greater mobility (Mr approx. 39000) were cut out and subjected to micro-sequencing by sequential Edman degradation on a 477A sequencer in tandem with a model 120A analyser (Applied Biosystems).
Enzyme assays.
Serine and alanine racemase activities of suitable dilutions of cytoplasmic and membrane fractions and of purified C-VanT were determined in a final volume of 30 µl. The assay mixture contained 100 mM Bistris propane pH 7·5, 10 mM L-serine or L-alanine and 10 µl of the diluted fraction as enzyme preparation. Mixtures were incubated at 37 °C for 40 min for the cytoplasmic and membrane preparations, and for 30 min at 37 °C and 42 °C for purified C-VanT. D-Amino acids produced by racemase activity were assayed using a D-amino acid oxidase assay (Messer & Reynolds, 1992 ), with D-serine or D-alanine as standards. The lower reliable limit of detection of D-Ser was 4 nmol min-1 (mg membrane protein)-1 and for D-Ala, 2 nmol min-1 (mg cytoplasmic protein)-1. Protein concentration was determined according to the method of Bradford (1976)
, with bovine serum albumin as standard.
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RESULTS |
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DISCUSSION |
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The alanine racemase activity of the purified His-tagged C-VanT was approximately 18% relative to its serine racemase activity (Table 4), similar to that of the membrane-bound enzyme (Table 2
). Alanine racemase activities of the serine racemases of brain and silkworms are considerably less (1·5% and 6% of the serine racemase activity respectively) (Wolosker et al., 1999a
; Uo et al., 1998
), indicating that the cytoplasmic domain of VanT is less selective than those of the eukaryotic enzymes. Recently, Wolosker et al. (1999b)
reported the cloning and sequencing of the pyridoxal-phosphate-dependent serine racemase from rat brain. Although the protein sequence does not exhibit significant homology with VanT it is likely that the mechanism of racemization of L-Ser is conserved in both proteins.
As VanT is likely to exist as a dimer (Arias et al., 1999 ) we explored the possibility of heterodimer formation between polypeptides of alanine racemase from E. coli and VanT. Cross-species heterodimers have been reported in different enzymes (Osterman et al., 1994
; Sun et al., 1992
; Greene et al., 1993
). Active cross-species heterodimers are usually not formed if the subunits share less than 80% identity in the dimer interface (Osterman et al., 1994
). An alignment between VanT and the alanine racemases of E. coli (Fig. 2
) revealed that the overall sequence identity is only 33%. Based on the crystal structure of Alr from B. stearothermophilus (Shaw et al., 1997
) we previously predicted that the residues from the other subunit of VanT involved in binding or catalysis could be Tyr597, Asp647 and Met648 (replaced by Gln314 in Alr) (Arias et al., 1999
), which are also conserved in the alanine racemases from E. coli (Fig. 2
). However, it is unlikely that the sequence identity between the subunits of these proteins at the dimer interface is as high as 80%. Also we did not find evidence of heterodimer formation based on N-terminal sequencing of different parts of the band of the purified His-tagged C-VanT from the SDS-PAGE gel or from sequencing of a protein with a similar Mr to that of alanine racemase that was eluted with the purified His-tagged C-VanT from the nickel column (Fig. 1
). The sequence obtained did not correspond to alanine racemases (instead it matched a glycerol dehydrogenase). One possible explanation for the lack of detection of subunits of alanine racemases is that being in a small proportion compared to the His-tagged C-VanT, they undergo blocking at the N-terminus and therefore become unavailable for Edman degradation. These results indicate that formation in vivo of heterodimers between subunits of alanine racemases of E. coli and the C-terminal domain of VanT is unlikely but if such heterodimers are present their proportion is likely to be very small compared to the formation of homodimers of C-VanT. Another possibility is that the topological organization of the transmembrane and cytoplasmic domains of VanT may play a role in amino acid selectivity for racemization. Investigation of this interaction is currently in progress.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 December 1999;
revised 31 March 2000;
accepted 7 April 2000.