GO was found to be active on various amines such as sarcosine, N-ethylglycine, glycine andD-amino acids, and to partially share substrate specificity with bothD-amino-acid oxidase DAAO,
Trang 1Overexpression of a recombinant wild-type and His-tagged Bacillus
Viviana Job, Gianluca Molla, Mirella S Pilone and Loredano Pollegioni
Department of Structural and Functional Biology, University of Insubria, Varese, Italy
We have cloned the gene coding for the Bacillus subtilis
glycine oxidase (GO), a new flavoprotein that oxidizes
gly-cine and sarcosine to the corresponding a-keto acid,
ammonia and hydrogen peroxide By inserting the DNA
encoding for GO into the multiple cloning site of the
expression vector pT7.7 we produced a recombinant plasmid
(pT7-GO) The pT7-GO encodes a fully active fusion protein
with six additional residues at the N-terminus of GO
(MARIRA) In BL21(DE3)pLysS Escherichia coli cells, and
under optimal isopropyl thio-b-D-galactoside induction
conditions, soluble and active chimeric GO was expressed up
to 1.14 U g)1of cell (and a fermentation yield of 3.82 UÆL)1
of fermentation broth) An N-terminal His-tagged protein
(HisGO) was also successfully expressed in E coli as a
soluble protein and a fully active holoenzyme HisGO
represents 3.9% of the total soluble protein content of the cell The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 UÆmg)1protein at 25°C and with a yield of 98% The characterization of the purified enzyme showed that GO is a homotetramer of 180 kDa with the spectral properties typical of flavoproteins GO exhibits good thermal stability, with a Tmof 46°C after 30 min incubation; its stability is maximal in the 7.0–8.5 pH range A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and onD-amino acids
Keywords: glycine oxidase; flavoprotein; oxidase; His-tagged protein; purification
Glycine oxidase (GO) is a flavoenzyme that was recently
discovered following the complete sequencing of the Bacillus
subtilisgenome [1] A preliminary investigation reported on
the expression of the yjbR gene product and on its partial
purification and characterization in Escherichia coli [2] GO
was found to be active on various amines such as sarcosine,
N-ethylglycine, glycine andD-amino acids, and to partially
share substrate specificity with bothD-amino-acid oxidase
(DAAO, EC 1.4.3.3) and sarcosine oxidase (SOX, EC
1.5.3.1) [2] GO catalyzes the oxidative deamination of
primary and secondary amines to give a-keto acids,
ammonia and hydrogen peroxide (L Pollegioni,
unpub-lished results) SOX catalyses the oxidative demethylation of
sarcosine (N-methylglycine) to form glycine and
formalde-hyde Similarly, DAAO catalyses the oxidative deamination
of neutral and (with a lower efficiency) basicD-amino acids
to give the corresponding a-keto acids and ammonia In
both cases, the reduced coenzyme is re-oxidized by
molecular oxygen to yield H2O2 Although DAAO and
SOX show a wide substrate tolerance, they do not efficiently
oxidize glycine
The present aim of this project is to elucidate the structure–function relationships in GO, with the ultimate goal of clarifying the modulation of the substrate specificity
in enzymes (GO, DAAO and SOX) that catalyse reactions
on similar compounds Although extensive information on the functional and structural characteristics of DAAO and SOX is available, there is little knowledge of GO properties and reactions There is also a biotechnological aspect to this project, as the stereoselective reaction catalysed by DAAO
is of considerable importance in biotechnology and indus-try, e.g the bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid [3,4] Industrial interest in the discovery of amino-acid oxidase activities with new, wider substrate specificity is increasing
Because of the relevance of this topic, we cloned the
B subtilisDNA that encodes GO and studied the overex-pression of this enzyme in E coli In this paper we report on two expression systems for obtaining fairly large quantities of
a recombinant chimeric GO from E coli cells and a quicker, high-yield procedure for the purification of this flavoenzyme
In addition, the physical and chemical characteristics, the activity on glycine and sarcosine, and the substrate specifi-city of the purified enzyme have been characterized
M A T E R I A L S A N D M E T H O D S Materials
Restriction enzymes and T4 DNA ligase were obtained from Roche Diagnostics Ampicillin, chloramphenicol, Luria–Bertani broth, o-dianisidine and horseradish peroxi-dase were purchased from Sigma DEAE-Sepharose Fast Flow, phenyl-Sepharose CL-4B, Superdex 200, HiTrap Che-lating and PD10 prepacked columns were from Amersham
Correspondence to L Pollegioni, Dipartimento di Biologia Strutturale
e Funzionale, Universita` degli Studi dell’Insubria via J.H Dunant 3,
21100 Varese, Italy Fax: + 332 421500, Tel.: + 332 421506,
E-mail: loredano.pollegioni@uninsubria.it
Abbreviations: GO, glycine oxidase; TSOX, heterotetrameric sarcosine
oxidase; MSOX, monomeric sarcosine oxidase; DAAO, D -amino-acid
oxidase; DASPO, D -aspartate oxidase; DMGDH, dimethylglycine
dehydrogenase; SDH, sarcosine dehydrogenase; PIPOX, pipecolate
oxidase; INT, iodonitrotetrazolium chloride.
(Received 23 October 2001, revised 7 January 2002, accepted 16
January 2002)
Trang 2Pharmacia Biotech and hydroxyapatite was from
Bio-Gel-HTP All purification steps were performed using an
A¨KTA-FPLC system (Amersham Pharmacia Biotech)
The plasmid DNA was extracted and purified from E coli
cells using the FlexiPrep Kit and the DNA was extracted
from the gel using the Sephaglass BandPrep Kit (Amersham
Pharmacia Biotech) The pT7.7A plasmid was from USB;
BL21(DE3)pLysS E coli cells were from Novagen Inc
Assay for GO activity
GO activity was assayed using a Hansatech oxygen
electrode equipped with a thermostat to measure the oxygen
consumption at pH 8.5 and 25°C with 10 mMsarcosine as
substrate One unit of GO is defined as the amount of
enzyme that converts 1 lmol of substrate (sarcosine or
oxygen) per minute at 25°C The pH effect on GO activity
and stability was determined using a multicomponent
buffer: 15 mM Tris, 15 mM sodium carbonate, 15 mM
phosphoric acid, 250 mMpotassium chloride and 1% (v/v)
glycerol The high [KCl] was used to buffer against minor
changes in ionic strength at different pH values These
buffers were adjusted to the appropriate pH with HCl or
KOH To assess the pH stability, activity was determined
30 min after incubation at the different pH values For
thermal inactivation, GO samples in 50 mM potassium
phosphate buffer, pH 7.0, containing 10% (v/v) glycerol
were incubated in a temperature range of 4–60°C Aliquots
were withdrawn after 30 min of incubation and assayed for
GO activity Protein concentration was measured using the
Bradford protein assay
Cloning ofB subtilis GO cDNA and DNA manipulation
The cloning and transformation techniques used were
essentially those described by Sambrook et al [5] Genomic
DNA from B subtilis cells strain B168 was a generous gift
of A Galizzi (Universita` di Pavia, Italy) PCR amplification
of the yjbR gene encoding GO was obtained using VentR
DNA polymerase (New England BioLabs) and the
fol-lowing oligonucleotides: YJBr-up (5¢-GCCATGAATTC
GCGCTATGAAAAGGCATTATGAAGCAGTGG-3¢)
derived from the 5¢ end and YJBr-down (5¢-CCGAT
GAATTCCATCATATCTGAACCGCCTCCTTGCG-3¢)
derived from the 3¢ end of nucleotide sequence of yjbR gene
(the sequence recognized by EcoRI restriction enzyme is
underlined) This amplification yielded a product of
1139 bp representing the entire GO gene The PCR product
was digested with the restriction enzyme EcoRI, isolated by
agarose gel electrophoresis and inserted in the unique EcoRI
site of the multiple cloning site of the expression vector
pT7.7 downstream of the T7 RNA polymerase promoter to
produce the recombinant plasmid pT7-GO The correct
orientation of the insert was checked by restriction digestion
with the enzyme HindIII Both strands of the resulting
plasmid were automatically sequenced: the nucleotide
sequence was identical to the known sequence of the yjbR
gene [1]
Expression and purification of GO inE coli
The pT7-GO expression plasmid was amplified in the E coli
strain JM109 and then transferred, for protein production,
to the host BL21(DE3)pLysS E coli strain Cells carrying the recombinant plasmid were grown at 37°C in Luria– Bertani, 2· Luria–Bertani, 2 · YT [5] or terrific broth media containing ampicillin (100 lgÆmL)1final concentra-tion) and chloramphenicol (34 lgÆmL)1 final concentra-tion), and induced at selected D600 values by adding isopropyl thio-b-D-galactoside After induction, the cells were grown at 30°C and collected at various times (from 1
to 24 h) by centrifugation Crude extracts were prepared by French Press lysis (three cycles at 1000 p.s.i.) of cell suspensions obtained by re-suspending the frozen cell paste with 50 mM sodium pyrophosphate buffer, pH 8.5, con-taining 5 mMEDTA, 0.2 lMFAD, 5 mM 2-mercaptoeth-anol, 0.7 lgÆmL)1pepstatin, 1 mMphenylmethanesulfonyl fluoride and 10 lgÆmL)1DNase (in a ratio of 2–3 mL of lysis buffer per gram of E coli cells) The insoluble fraction
of the lysate was removed by centrifugation at 39 000 g for
40 min at 4°C
The cell homogenate, obtained by French Press lysis of
34 g of E coli cells under the conditions reported above, was precipitated with ammonium sulfate at 30% of saturation (164 gÆL)1) The supernantant was then brought
to 45% of saturation (86 gÆL)1) After centrifugation, the protein pellet was re-suspended and dialysed against 50 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 10% glycerol and 5 mM 2-mercaptoethanol (buffer A) After dialysis and centrifugation, the enzyme solution was applied
to a DEAE-Sepharose Fast-Flow column (1.6· 21 cm) and eluted with a 10–20% linear gradient using buffer A to which 1M NaCl was added Fractions containing GO activity were pooled and concentrated using an Amicon cell concentrator equipped with a YM30 membrane The sample was then loaded onto a hydroxyapatite column (1.6· 12 cm) and GO eluted, using a 50 mM potassium phosphate buffer, pH 6.5, 2 mMEDTA and 10% glycerol The fractions containing GO activity were pooled and the
pH was adjusted with sodium carbonate to pH 7.5 After the addition of 0.5Msodium chloride (final concentration) the sample was applied to a phenyl-Sepharose CL-4B column (1.6· 22 cm) equilibrated in 50 mM potassium phosphate buffer, pH 7.5, 0.5 M sodium chloride, 2 mM EDTA, and 5 mM2-mercaptoethanol The bound enzyme was subsequently eluted with 50 mMpotassium phosphate,
pH 7.5, 5 mM 2-mercaptoethanol and 2 mM EDTA The fractions containing GO activity were concentrated and stored at)20 °C
HisGO preparation, expression and purification The GO DNA obtained by EcoRI digestion of the original pT7-GO plasmid (see above) was inserted into the EcoRI site of the pT7-DBam/Hind plasmid [6] The resulting recombinant plasmid, defined as pT7-HisGO, encodes for
an additional N-terminal sequence containing one methi-onine and six histidine residues (Fig 1) The correct insertion of the GO gene was checked by digestion with the restriction enzymes NdeI and BamHI/ScaI and by a PCR reaction, using the XbaI [6] and YJBr-down oligonu-cleotides as a primer
The pT7-HisGO expression plasmid was transferred
to the host BL21(DE3)pLysS E coli strain for protein production Recombinant cells were grown at 37°C in
2· Luria–Bertani medium containing ampicillin and
Trang 3chloramphenicol (100 lgÆmL)1and 34 lgÆmL)1final
con-centration, respectively) and induced at D600 ¼ 0.8 by
adding isopropyl thio-b-D-galactoside at a final
concentra-tion of 1 mM The cells were then grown at 30°C and
collected after 24 h by centrifugation Crude extracts
(prepared as described above) were added of 1 M NaCl
and 1 mM imidazole (all final concentrations) to improve
the interaction specificity in the affinity chromatography
step The enzyme solution was then applied to a HiTrap
chelating affinity column (5 mL) equilibrated with 50 mM
sodium pyrophosphate buffer, pH 7.2, containing 1 M
NaCl, 20 mM imidazole and 5% glycerol, using an
A¨KTA-FPLC system The bound enzyme was eluted with
50 mM sodium pyrophosphate buffer, pH 7.2, containing
500 mM imidazole and 5% glycerol (elution buffer) The
fractions containing GO activity were pooled and loaded on
a PD10 column equilibrated with 50 mM sodium
pyro-phosphate buffer, pH 8.5, containing 2 mMEDTA, 5 mM
2-mercaptoethanol and 10% glycerol
Substrate specificity
Two different methods were used to investigate the substrate
specificity of GO
Activity stain using iodonitrotetrazolium chloride (INT)
on protein samples separated by native PAGE After the
electrophoretic separation, each single lane was incubated
for 2 h in the dark at 37°C in a solution containing 75 mM
sodium pyrophosphate, pH 8.5, 100 mM substrate, 5 lM
FAD and 0.1 mgÆmL)1 INT (dissolved in pure ethanol)
The activity was revealed as a pink band on the gel in the
position corresponding to the GO
Spectrophotometric determination of GO activity
meas-uring the hydrogen peroxide produced in the presence of
different substrates using a 96-well ELISA plate Each assay well has 200 lL of a solution containing 10 mMor
90 mM substrate, 0.32 mgÆmL)1 o-dianisidine and
10 UÆmL)1 of horseradish peroxidase in 90 mM sodium pyrophosphate, pH 8.5 The increase in absorbance at
440 nm was followed using a Metertech 960 spectropho-tometer (extinction coefficient of o-dianisidine product was
13 mM )1Æcm)1)
Western blot analysis, N-terminal sequencing and sequence comparison
Western blot analysis was performed on total E coli and crude protein extracts Proteins were separated on 12% SDS/PAGE [7] and transferred electrophoretically to nitrocellulose membranes (Millipore) [8] GO was detected
by immunostaining using rabbit anti-GO Ig and visual-ized using anti-(rabbit IgG) Ig alkaline phosphatase conjugate with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue-tetrazolium chloride The anti-GO Ig were raised in rabbit with 0.5 mg of pure GO as antigen according to a standard protocol (Davids Biotechnologie, Germany) The N-terminal sequence of the recombinant
GO was determined on a purified soluble protein sample using an automated protein sequencer (Procise Model
492, Applied Biosystems) The BLAST program (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi) [9] was used to search for proteins showing sequence similarity Multiple sequence alignments were performed with the CLUSTALW program (http://npsa-pbil.ibcp.fr/cgi-bin/align_clustalw.pl) [10]
R E S U L T S Cloning ofB subtilis GO DNA and protein sequence comparison
To clone the gene encoding the B subtilis GO, the genomic DNA was amplified by PCR using the oligonucleotides derived from the sequence of the yjbR gene of GO from
B subtilis [1] Currently, there are three proteins in the nonredundant Protein Data Bank that have been classified
as GO In addition to the enzyme from B subtilis, the proteins from Bacillus halodurans and Thermoplasma volcanium (accession nos BAB05153 and NP_111169, respectively) have been also included in the database The overall sequence identity with B subtilis GO is modest (27% and 22%, respectively), although a higher conserva-tion is evident at their N-termini (containing the Rossman fold fingerprint motif GXGXXG, which is involved in binding of the ADP moiety of FAD) [11] and for the 70 residues at their C-termini (for this latter region a 31–40%
of identity and a 53–61% of sequence similarity has been calculated)
The results of comparison of the amino-acid sequence of
B subtilis GO with the sequences available in the Data Bank are reported in Table 1 The greatest similarity was found between GO and the b subunit of heterotetrameric sarcosine oxidase (TSOX), sarcosine dehydrogenase (SDH) and dimethylglycine dehydrogenase (DMGDH) In all cases, the N-terminal region corresponds to the flavin-binding domain and shows the highest sequence conserva-tion (32–48% of identity when only the starting 50 residues
Fig 1 Physical map of the Bacillus subtilis GO expression plasmid
pT7-HisGO The 1.13 kb of GO cDNA was inserted into the EcoRI
site of the MCS of pT7-DBam/Hind plasmid [6] The original ATG
starting codon is shown in bold and the ATG codon corresponding to
the Met1 of the wild-type GO in italic.
Trang 4are considered) A considerable homology was also
observed with the sequences of monomeric sarcosine
oxidases (MSOX), pipecolate oxidase (PIPOX), DAAO
andD-aspartate oxidase (DASPO) PIPOX is a mammalian
enzyme that plays a significant role in brain metabolism,
whereL-pipecolate acts as a neuromodulator DAAO has
been proposed to regulate the levels ofD-serine in brain, a
D-amino acid that modulates NMDA neurotransmission A partial sequence comparison between GO and SOX or DAAO proteins [12,13] of the regions containing the highly conserved residues that play a key role in catalysis is reported in Fig 2A,B
Table 1 BLAST results of search for identity and homology with B subtilis GO The percentages refer to the 369 amino acids of GO The homology score is considered the sum of identical and strongly similar amino acids The proteins identified by the code ÔNP_Õ have been recognized following the complete sequence of the genome of the corresponding organism; therefore, their activity was never proven TSOX, b subunit of hetero-tetrameric sarcosine oxidase.
Entry code Organism Protein Identity (%) Homology (%) BAB05153 Bacillus halodurans GO 27 51.8
NP_111169 Thermoplasma volcanium GO 21.7 48.0
NP_107418 Mesorhizobium loti TSOX 24.4 45.5
NP_126006 Pyrococcus abyssi TSOX 26.8 49.9
P40875 Corynebacterium sp P-1 TSOX 23.8 48.8
P40859 Bacillus sp B-0618 MSOX 20.6 43.4
P80324 Rhodotorula gracilis DAAO 18.4 38
NP_106044 Mesorhizobium loti SDH 27.6 49.6
NP_057602 Homo sapiens PIPOX 21.4 46.9
Fig 2 Details of the multiple sequence alignment for GO from B subtilis with sequences of SOX (A) and DAAO (B) and comparison of the 3D structure of the active site with those of other enzymes (C) Residues marked with (*) indicate identity (:) indicate strongly similar, and (.) indicate weakly similar The boxes identify the amino acids present in the active site of MSOX [12] and of DAAO [15,16] (C) Comparison of the active site
of the 3D-structure model of GO with the active site of MSOX from Bacillus subtilis (right) [12] and DAAO from Rhodotorula gracilis (left) [15].
Trang 5Using the Swiss-Model software at theEXPASYwebsite
[14], a model of the 3D structure of GO has been predicted
using its protein sequence and the 3D coordinates of
Rhodotorula gracilis DAAO as template [15] Due to the
limited sequence homology between these two enzymes, the
GO model only extends from Y240 to G313 A comparison
of the active sites of MSOX and DAAO with the active site
of the model derived for GO (Fig 2C) confirms that Y246 is
highly conserved and is located at a position resembling that
of Y223 in DAAO and Y254 in MSOX Even the arginine
residue (R285 in yeast DAAO) which electrostatically
interacts with the a-carboxylate of the D-amino acid in
DAAO [15,16] is conserved in GO (R302, see Fig 2C) On
the other hand, and according to the sequence comparison,
M261 in GO appears to be located at the position occupied
by Y238 and H269 in DAAO and MSOX, respectively This
second active site residue, which is involved in the
recog-nition/binding of the substrate in both MSOX and DAAO
[12,15], is not conserved in GO and probably represents a
key element for defining its substrate specificity
The limited (sequence) similarity/homology between GO,
DAAO, MSOX and TSOX is also confirmed by the results
obtained in Western blot experiments using monospecific
anti-DAAO [17] and anti-GO Ig The antibodies raised
against pure B subtilis GO did not recognize DAAO and
MSOX (a faint band is only observed when a large amount
of TSOX is used, data not shown) Analogously,
anti-DAAO IgG did not recognize GO, MSOX or TSOX
Expression ofB subtilis GO gene in E coli
and GO purification
The DNA encoding for GO was inserted in the EcoRI site
of the multiple cloning site of the expression vector pT7.7
downstream of the T7 RNA polymerase promoter to
produce the recombinant plasmid pT7-GO Because of the
presence of an ATG codon upstream of the cloning site, this
procedure produces a fusion protein; six additional residues
are added at the N-terminus of the protein before the
original starting methionine The new ATG starting codon
is positioned 8 bp downstream from the ribosomal binding
site The recombinant plasmid pT7-GO was used to
transform BL21(DE3)pLysS E coli cells A significant
increase in GO synthesis was observed immediately after
the addition of 1 mMisopropyl thio-b-D-galactoside in cell
cultures transformed with the pT7-GO expression vector
and grown on Luria–Bertani medium, as indicated by native PAGE and GO activity staining The highest level of GO expression and GO specific activity was obtained 24 h after induction in the mid-log growth phase of a culture grown on Luria–Bertani medium No activity was detected in E coli extracts from cells carrying the vector without the 1.13-kb
GO DNA (data not shown) The GO expression depends
on the isopropyl thio-b-D-galactoside concentration: West-ern blot experiments show that the maximal amount of immunoreactive GO in soluble crude extract, as well as in the total cell, was achieved at an isopropyl thio-b-D -galactoside concentration of 0.5 mM Analogously, the higher GO fermentation yield (in terms of GO units per gram of cell paste and GO specific activity) was obtained at the same concentration of inducer (data not shown) A comparison of the amount of immunoreactive GO in the total cell with the amount of GO in the crude extract on Western blot indicates that GO is almost totally expressed in
a soluble form Under the best conditions, the GO expression reached 1 UÆg)1cell and 4 UÆL)1of fermen-tation broth (with a GO specific activity in the crude extract
of 0.006 UÆmg)1protein) GO accumulated to 1% of total
E colisoluble proteins in the crude extract
Recombinant GO was purified from E coli cells by precipitation with ammonium sulfate twice and then a three-step chromatography procedure (see Materials and methods) About 13 mg of enzyme (90% homogenous) were obtained starting from 34 g of cell paste (Table 2) The total yield of the purification was about 20% The N-terminal sequence of the first eight amino acids of the recombinant GO was determined This sequence is unique and was identical to that inferred from the nucleotide sequence [1]; protein sequencing of 0.2 nmol of the purified enzyme resulted in the ARIRAMKR sequence, confirming the presence of five of the six additional residues at the N-terminus of recombinant chimeric GO (the starting methionine is not present)
The recombinant GO produced in E coli was purified
as a holoenzyme with spectral properties typical of the flavin-containing oxidases (absorbance maxima at 457 nm and 376 nm and a A274/A456 ¼ 9) Native GO is a 180-kDa homotetramer, as determined by SDS/PAGE (46.6 ± 1.3 kDa) and gel permeation chromatography on
a Superdex 200 column (187.6 ± 2.3 kDa) The recombin-ant enzyme catalysed the oxidation of sarcosine and glycine (specific activity of 0.57 and 0.43 UÆmg)1protein, at 25°C)
Table 2 Purification of recombinant B subtilis glycine oxidase from BL21(DE3)pLysS E coli cells containing the pT7-GO (top) and pT7-HisGO (bottom) plasmid Starting material was 34 g of E coli cell paste obtained from a 10-L fermentation (GO) and 38 g of E coli cell paste obtained from
a 6-l fermentation (HisGO).
Step
GO activity (U)
Protein content (mg)
Specific activity (UÆmg protein)1)
Purification index
Yield (%) GO
(NH 4 ) 2 SO 4 precipitation 25.0 2984 0.008 1.4 65.4
Phenyl-Sepharose 7.5 13.1 0.568 98.0 19.5 HisGO
Trang 6Addition of exogenous FAD or FMN to the assay mixture
did not increase enzyme activity, indicating a tight binding
of the coenzyme to the protein moiety
Preparation, overexpression and purification
of the chimeric His-tagged GO
The scheme of the strategy devised for obtaining a plasmid
coding for the chimeric His-tagged GO is reported in the
Materials and methods section; the map of the resulting
expression vector pT7-HisGO is given in Fig 1 In these
experiments the conditions for the expression of GO in
E coliBL21(DE3)pLysS cells were optimized Cells were
grown at 37°C and induced with 1 mMisopropyl thio-b-D
-galactoside at an D600 ¼ 0.8; the growth temperature was
then decreased to 30°C and the cells were harvested 24 h
after induction The protein was overexpressed under the
reported conditions and was totally soluble and thus
recovered in the crude extract In addition, the chimeric
HisGO was entirely present as an active holoenzyme
Therefore, the addition of exogenous FAD or FMN to the
assay mixture did not increase the activity at all The enzyme
expression was about 1 UÆg)1 of wet weight ( 2.1 mg
GO/g of wet weight cell and 4% of total E coli soluble
proteins in the crude extract) and the fermentation yield was
14.4 UÆL)1per culture
The crude extract from induced E coli cells was loaded
onto a HiTrap chelating affinity column following the
addition of 1Msodium chloride and 1 mMimidazole (all
final concentrations) Under the elution conditions we
employed (see Materials and methods), the engineered GO
was selectively eluted as a single resolved peak (data not
shown) The final preparation was 95% homogeneous The
sequence analysis (MHHHHHHMARIRA) of the purified
enzyme confirmed the presence of the basic additional
sequence (the starting methionine plus the six histidine tag
plus the additional six residues of the chimeric protein) at
the N-terminus, which clearly does not impair either the
enzymatic activity or the coenzyme binding
The purification of the His-tagged chimeric GO and, for
comparison, that of the wild-type enzyme is reported in
Table 2 From this table it is apparent that the purification
procedure of the engineered GO is far less time-consuming
(the final GO preparation is obtained in few hours starting
from the E coli cell paste) Remarkably, and due to the fast
procedure used, we obtained a fully active holoenzyme with
a specific activity that was twofold higher than that of the
wild-type recombinant enzyme (1.06 vs 0.57 UÆmg)1
pro-tein) In addition, the total recovered enzyme activity
obtained starting from a similar amount of E coli cell paste
are higher for the HisGO than for the wild-type enzyme
Properties of the His-tagged chimeric GO
The molecular mass of HisGO, as determined by SDS/
PAGE, is slightly higher than the value calculated from
the amino-acid sequence (49.4 ± 1.1 kDa vs 42.66 kDa,
respectively) Under native conditions, the molecular mass
of the His-tagged GO is 166.4 ± 11.1 kDa, as determined
by gel permeation chromatography The elution volume
(Rt ¼ 13.4 ± 0.2 mL) is not dependent on the protein
concentration in the range 0.01–12 mgÆmL)1 The result is
consistent with the presence in solution of a stable
homotetramer The theoretical isoelectric points of GO and HisGO are 6.14 and 6.34, respectively However, we were not able to determine this under any of the present experimental conditions due to the instability of the protein
at a pH < 7 (see below) The purified holoenzyme shows the typical absorbance spectrum of FAD-containing proteins, with two well-resolved peaks in the visible region (at 458 nm and 378 nm, see Fig 3) Anaerobic addition of
an excess of glycine resulted instantaneously in the forma-tion of reduced enzyme flavin, as shown by its spectrum with a maximum at 346 nm (Fig 3) The spectral properties
of oxidized and reduced recombinant GO are significantly different from those previously reported, e.g the reported flavin reduction following addition of 10 mM sarcosine resulted in a 20% decrease of the 455 nm peak [2], which
is not compatible with full flavin reduction
Plotting GO activity (on sarcosine as substrate and using both GO and HisGO) over the temperature range 15–60°C shows an increase in activity with no evidence for any plateau or decrease up to 60°C (Fig 4A) HisGO shows good temperature stability in the range 4–35°C when the samples are incubated for 30 min at pH 7.0 When the temperature is increased to above 35°C, however, a sharp decrease in enzyme stability is evident (Fig 4A): a Tmof
46°C was seen The activity is maximal at about pH 10, but falls off rapidly above pH 10, probably due to instability (Fig 4B) The stability is maximal in the 7–8.5 pH range Below pH 6.5 and above pH 9.5, a considerable decrease in stability was observed (Fig 4B) These profiles were iden-tical when GO was used instead of HisGO (data not shown) Both GO and HisGO enzymes are stable for months when stored at)80 °C and at a pH ¼ 7.5 Substrate specificity
Purified GO shows a similar specific activity on sarcosine and glycine as substrates (1.06 and 1.00 UÆmg)1 protein, respectively) Because of the sequence similarity of GO with
Fig 3 Absorbance spectrum of the purified HisGO ( 12 l M ) in the oxidized form (1) and in the reduced form (2), as obtained under anaerobic conditions by the addition of 20 m M glycine in 50 m M potas-sium phosphate buffer pH 7.0, 5 m M 2-mercaptoethanol, 2 m M EDTA and 10% glycerol.
Trang 7a number of flavoenzymes (see Table 1), the substrate
specificity of the reaction catalysed by GO has been
investigated using several compounds that are known to
be substrates of DAAO, DASPO, SOX, DMGDH and
PIPOX Rapid screening was performed by measuring the
increase in absorbance at 440 nm following the horseradish
peroxidase assay coupled with o-dianisidine using a 96-well
ELISA plate The results, reported as a percentage with
respect to the absorbance change observed with sarcosine as
100%, are shown in Fig 5 From this it is evident that GO
possesses a wide substrate specificity In addition to
sarcosine and glycine, even N-ethylglycine, ethylglycine
ester,D-alanine,D-2-aminobutyrate,D-proline,D-pipecolate
and N-methyl-D-alanine are good substrates; here, a lower activity is observed on branched and polarD-amino acids (e.g.D-Val,D-Ile,D-Leu,D-His, andD-Arg) GO is strictly stereospecific as it only catalyses the oxidation of the
D-isomer, as further demonstrated by the INT staining assay reported in Fig 5, inset
A comparison of the activity measured using a similar amount (5 mU) of GO, DAAO and MSOX on various substrates showed that GO and DAAO oxidizeD-proline,
D-alanine and D-2-aminobutyrate with similar relative efficiencies Analogously, GO and MSOX show a fairly similar activity on sarcosine and N-ethylglycine In contrast,
an appreciable activity on glycine, glycine-ester and
D-pipecolic acid was only observed using GO Interestingly,
D-proline is the only amino acid in which the a-amino group
is involved in a covalent bond with the side chain and it is the only compound that was oxidized by all three enzymes used It is also the only D-amino acid that is oxidized efficiently by both DAAO andD-aspartate oxidase [18]
C O N C L U S I O N S The present data demonstrate the successful overexpression
of a chimeric B subtilis GO in E coli (up to 3.9% of total soluble protein) using two different expression systems In particular, we produced a recombinant GO containing a polyhistidine tag at the N-terminus, that can be efficiently purified in a single step by metal-chelate chromatography, producing a stable holoenzyme with a high specific activity and a 98% yield quickly and simply Its properties (aggregation state, absorbance spectrum and stability) are identical to those of native GO, but the specific activity is twofold higher The spectral properties of GO in the oxidized and reduced form (Fig 3) unambiguously identify
it as a flavoenzyme A comparison of the expression level and purification yield obtained with the previous procedure [2] is not feasible because no quantitative data have been previously reported
Sequence comparison and Western blot analyses show that GO is not highly similar to any known flavo-enzyme, although the observed sequence homology and
Fig 5 Assay of GO activity on a number of compounds using the horseradish peroxidase assay and o-dianisidine The values are reported as a percentage of the absorbance change at 440 nm obtained with sarcosine (referred as 100%) after 3 h of incubation at pH 8.5, pH 5.8 (a) or pH 9.7 (b) Inset: assay of GO activity on various compounds using the activity stain with INT on enzyme samples (6 lg) separated by native PAGE in the presence of: (1) sarcosine; (2) glycine; (3) N-ethylglycine; (4) N,N-dimethylglycine; (5) D -alanine; (6) L -alanine; (7) D -proline; (8) L -proline; (9) -valine; (10) -valine, as substrate.
Fig 4 Temperature–activity and temperature stability profiles (A) and
pH–activity and pH stability profiles of HisGO (B) Enzyme activity (d)
was assayed in the temperature range 15–60 °C For thermostability
(s) enzyme samples were incubated at the indicated temperature for
30 min in 50 m M potassium phosphate, pH 7.0, 10% glycerol and then
assayed using the O 2 consumption assay at 25 °C Data are expressed
as per cent of enzyme activity in the standard assay; the lines through
the data points have been obtained by smooth fitting SEM is the
average of three determinations (B) pH–activity and pH stability
profiles of HisGO Enzyme activity (d) was assayed in the pH range
4.8–12 For thermostability (s) enzyme samples were incubated at the
indicated pH for 30 min in the multicomponent buffer (see Materials
and methods) and then assayed using the standard O 2 consumption
assay at 25 °C Data are expressed as per cent of enzyme activity in the
standard assay; the lines through the data points have been obtained
by smooth fitting The SEM is the average of three determinations.
Trang 8conservation of active site residues suggest that GO would
exhibit significant structural similarity with enzymes acting
on sarcosine or sarcosine analogues (monomeric and
tetra-meric sarcosine oxidases and sarcosine and dimethylglycine
dehydrogenases), onD-amino acids (DAAO and DASPO)
and on pipecolic acid (PIPOX) (see Table 1) The metabolic
role of GO in B subtilis and its physiological substrates are
unknown Its substrate specificity partially overlaps with
that of DAAO and SOX and indicates an oxidative role of
this oxidase in B subtilis on a wide range of compounds
The cloning and overexpression of the B subtilis GO also
provides the basis for kinetic, site-directed mutagenesis and
structural studies and, thus, identification of the underlying
structure of the catalytic properties and substrate specificity
of this novel flavoenzyme This may, in turn, make it possible
to engineer the GO activity for use as a biocatalyst In fact,
the biotechnological applications for DAAO-catalysed
reactions (a world market of 400 million USD for the
production of 7-aminocephalosporanic acid alone has been
estimated; reviewed in [4]) require the production of a large
amount of this flavoenzyme as well as of amino-acid
oxi-dase activities with different or altered substrate specificities
A C K N O W L E D G E M E N T S
This work was supported by grants from Fondo di Ateneo per la
Ricerca (2000) to L P., and from ÔProgetto Giovani RicercatoriÕ
(University of Insubria) to V J.
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