A series of UV absorption spectra obtained in stopped-flow experiments using the wild-type and mutant enzymes revealed isosbestic points indicative of apparently perfect reactions, which
Trang 1Screening for an improved GTP cyclohydrolase II mutant
Martin Lehmann1, Simone Degen1, Hans-Peter Hohmann1, Markus Wyss1, Adelbert Bacher2and Nicholas Schramek2
1 DSM Nutritional Products Ltd., Basel, Switzerland
2 Lehrstuhl fu¨r Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstr, Garching, Germany
Introduction
More than 3000 metric tons of vitamin B2 (riboflavin;
6) are produced per year for use in human nutrition,
animal husbandry and as a food colorant In recent
years, efficient fermentation processes have replaced
chemical synthesis for manufacturing the vitamin [1,2]
The biosynthetic pathway of riboflavin has been
studied in considerable detail [3–6] Briefly, GTP is
converted into
2,5-diamino-6-ribosylamino-4(3H)-pyri-midinone 5¢-phosphate (2) by the catalytic action of
GTP cyclohydrolase II (Fig 1) [7] The product is
transformed into
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (3) by a sequence of side-chain
reduc-tion, deamination and dephosphorylation Condensa-tion of 3 with 3,4-dihydroxy-2-butanone 4-phosphate (4) results in the production of 6,7-dimethyl-8-ribityl-lumazine (5) [8,9] An unusual dismutation catalyzed
by riboflavin synthase converts the lumazine derivative into an equimolar mixture of riboflavin (6) and the pyrimidine 3 which is re-utilized by the lumazine synthase [10–13] With the exception of the elusive phosphatase, all enzymes of the pathway have been studied at least in some detail
The enzymes of the riboflavin pathway are generally characterized by low catalytic rates This is not surprising
Keywords
biotechnology; directed evolution; GTP
cyclohydrolase; riboflavin biosynthesis;
vitamin B 2 production
Correspondence
N Schramek, Lehrstuhl fu¨r Biochemie,
Technische Universita¨t Mu¨nchen,
Lichtenbergstr 4, D-85747 Garching,
Germany
Tel: +49 089 289 13336
Fax: +49 089 289 13363
E-Mail: nicholas.schramek@ch.tum.de
(Received 16 March 2009, Revised 24 May
2009, accepted 28 May 2009)
doi:10.1111/j.1742-4658.2009.07118.x
GTP cyclohydrolase II catalyzes the first dedicated step in the biosynthesis
of riboflavin and appears to be a limiting factor for the production of the vitamin by recombinant Bacillus subtilis overproducer strains Using error-prone PCR amplification, we generated a library of the B subtilis ribA gene selectively mutated in the GTP cyclohydrolase II domain The ratio of the GTP cyclohydrolase II to 3,4-dihydroxy-2-butanone synthase activities
of the mutant proteins was measured A mutant designated Construct E, carrying seven point mutations, showed a two-fold increase in GTP cyclo-hydrolase II activity and a four-fold increase in the Kmvalue with GTP as the substrate Using the analog 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5¢-triphosphate as the substrate, the mutant showed a rate enhancement by a factor of about two and an increase in the Kmvalue
by a factor of about 5 A series of UV absorption spectra obtained in stopped-flow experiments using the wild-type and mutant enzymes revealed isosbestic points indicative of apparently perfect reactions, which were simi-lar to the findings obtained with GTP cyclohydrolase II of Escherichia coli Initial burst velocities obtained for the mutant and wild-type proteins were similar The data suggest that the mutations present in Construct E are jointly conducive to the acceleration of a late step in the reaction trajec-tory, most probably the release of product from the enzyme
Abbreviation
DHB, 3,4-dihydroxy-2-butanone.
Trang 2in light of the small amounts of the trace metabolite
that are required for metabolism and growth These
low rates constitute a problem for the further
improve-ment of riboflavin ferimprove-mentation processes
Studies on a riboflavin producer strain of
Bacil-lus subtilisshowed a significant increase in productivity
following the insertion of an additional gene copy of
ribA, suggesting that this enzyme constitutes a
bottle-neck in the pathway [14]
The complex reaction mechanism of GTP
cyclohy-drolase II has been studied using spectroscopic and
kinetic methods Notably, the enzyme catalyzes the
release of C-8 from the imidazole moiety of GTP as
formate and also the release of inorganic diphosphate
from the ribose side-chain (Fig 2) As a side reaction,
a fraction of the substrate, GTP, is converted into
GMP (11) by release of pyrophosphate without
con-comitant ring opening Kinetic studies suggested that
the first reaction step of the enzyme-catalyzed
trajec-tory is the covalent guanylation of the enzyme under
release of pyrophosphate [12] Carbon 8 of the purine
system of intermediate 8 is then hydrolytically released,
and the reaction is terminated by hydrolysis of the
phosphodiester bond between the covalently bound
intermediate 9 and the protein
This article describes studies directed at an increase
in the overall reaction rate of GTP cyclohydrolase II
Results
Whereas most enzymes of the riboflavin biosynthetic pathway have low catalytic rates, the activity of GTP cyclohydrolase II appears to be rate limiting for the overall productivity of a recombinant B subtilis strain [14] Both initial steps of the convergent riboflavin bio-synthetic pathway are catalyzed in B subtilis by the bifunctional RibA protein comprising a GTP cyclohy-drolase II and a 3,4-dihydroxy-2-butanone 4-phosphate domain on the same subunit In order to increase selec-tively the GTP cyclohydrolase II activity, the gene seg-ment specifying the cognate protein domain was subjected to in vitro mutagenesis by error-prone PCR (on average, two to five mutations per gene), and the resulting amplificates were ligated to the gene segment specifying the 3,4-dihydroxy-2-butanone 4-phosphate domain (that had not been subjected to mutagenesis) The resulting, mutated genes were ligated into the expression plasmid pQE60 and transformed into an Escherichia coli strain carrying a ribA) mutation (Fig 3) Growth occurred only if the mutated ribA
C
D
E
Fig 1 Pathway of riboflavin biosynthesis (A) GTP cyclohydrolase II (B) Sequence of deaminase, reductase and phosphatase (C) 3,4-Dihydroxy-2-butanone 4-phosphate synthase (D) 6,7-Dimethyl-8-ribityllumazin synthase (E) Riboflavin synthase.
Trang 3gene specified a recombinant protein that retained
sig-nificant GTP cyclohydrolase II activity The library of
recombinant E coli strains afforded a library of mutant
proteins that was assayed for GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase activities Mutant proteins with a relative increase in GTP cyclohydrolase II activity (compared with the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity) were purified; purification was facilitated by the pres-ence of a polyhistidine tag at the N-terminus
A total of 3300 recombinant E coli strains were screened and provided nine candidate strains with apparent enhancements in GTP cyclohydrolase II activ-ity; the largest enhancements observed were in the range of 1.5-fold Combination of the gene mutations
of the most improved mutant genes A#1 G2 (T203S, A290T, A296T) and A#4 C9 (K195T, V264A, V275A, K397E) did not result in a further improved mutant Numbering of the amino acid residues included the 14 amino acids of the His-tag The original start methio-nine of RibA became amino acid residue 15 The neutral mutation, T203S, of A#1 G2, was removed, and mutation Y210C, which was found in another mutant of the library, was introduced in return By SDS gel chromatography it became apparent that the
Fig 2 Hypothetical reaction mechanism of GTP cyclohydrolase II
[13].
mrgshhhhhhgidh
Fig 3 Generation of the cyclohydrolase II mutant library The DHB synthase and cyclohydrolase II domains were separately amplified
by PCR to permit the integration of random mutations only into the cyclohydrolase II domain Afterwards, the two PCR products were combined by a third PCR, digested by EcoRI and BamHI, and trans-formed into the cyclohydrolase II-deficient E coli strain Rib7 (pREP4).
Trang 4mutation reduced the susceptibility to proteolytic
cleveage into two typical fragments of the RibA
wild-type protein The new mutant (Y210C, A290T, A296T)
was used as template for a second cycle of mutagenesis
and selection (10 000 mutants) It afforded 351 novel
candidate strains After rescreening, 10 mutant proteins
were purified and characterized, and their effective
mutations were determined The best combinations of
the newly found mutations resulted in Constructs C
(Y210C, A290T, Q293R, A296T, K322R, M361I) and
E (Y210C, A290T, Q293R, A296T, K322R, F339Y,
M361I), which were selected for more detailed kinetic
studies
Steady-state kinetic experiments were conducted at
pH 8.5 and 30C The reaction was monitored
photo-metrically at 310 nm Figure 4 shows experiments
using GTP as substrate Experimental data points
showed good agreement with the Michaelis–Menten
approximation over a wide range of substrate
concen-trations (0.017–1.7 mm) The Vmax value of Construct
C exceeded that of the wild-type protein by a factor of
1.9 Constructs C and E both showed Km values that
were increased substantially, by a factor in the range
of three- to four-fold, compared with that of the
wild-type protein Notably, the steady-state parameters of
the B subtilis wild-type protein were similar to those
of GTP cyclohydrolase II of E coli that has been
stud-ied previously in some detail [11,15]
Steady-state kinetic experiments were also performed
with the reaction intermediate 10 (prepared from GTP
by enzymatic treatment with a mutated GTP
cyclohy-drolase I, as described in Ref [16]) Experiments were
monitored photometrically at 310 nm (Fig 5) The
maximum rate observed with Construct E was again increased by a factor of about 2 Again, the mutated Constructs C and E showed markedly increased Km values (Table 1)
It appears likely that the hydrolytic opening of the imidazole ring of GTP has the highest Gibbs free energy barrier of all partial reactions in the GTP cyclohydrolase II trajectory However, the comparative steady-state analysis using the natural substrate, GTP, and the ring-opened reaction intermediate 10, suggests that the increase in the overall rate constants observed with the mutated proteins is not caused by a lowering
of that free energy barrier
Previously, we studied GTP cyclohydrolase II of
E coliusing presteady-state kinetic analysis [13] Unex-pectedly, those experiments had suggested a relatively slow formation of the phosphoguanosyl derivative 7 under release of pyrophosphate That covalently bound moiety appeared to undergo rapid hydrolytic release of formate from the imidazole ring and⁄ or hydrolytic cleavage of the phosphodiester bond It was, in fact, a
Fig 4 Steady-state kinetics of GTP cyclohydrolase II from
Bacil-lus subtilis, using GTP as the substrate Symbols represent the
experimental data Lines represent the Michaelis–Menten
approxi-mation (—, wild-type; —, Construct E; ÆÆÆÆ, Construct C).
Fig 5 Steady-state kinetics of GTP cyclohydrolase II from Bacil-lus subtilis using 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyri-midinone 5¢-triphosphate (Compound 10) as the substrate Symbols represent the experimental data (310 nm) Lines represent the Michaelis–Menten approximation (—, wild-type; - - -, Construct E).
Table 1 Kinetic properties of different GTP cyclohydrolase II proteins from Bacillus subtilis.
GTP as substrate
Compound
10 as substrate
kcat(min)1) Km(l M ) kcat(min)1) Km(l M )
Construct E 4.3 ± 0.04 44 ± 2 6.0 ± 0.3 122 ± 20 Construct C 3.9 ± 0.04 49 ± 3 5.4 ± 0.1 79 ± 8
Trang 5surprising finding that the opening of the imidazole ring
was not in any way rate-limiting, despite the relatively
high free-energy barrier of that reaction step
We have now conducted similar stopped-flow experi-ments with the wild-type and mutant enzymes of
B subtilis By comparison with the earlier study, the present experiments were hampered by the tendency of the B subtilis enzymes to form precipitates after the addition of GTP that were conducive to corruption of the photometric signal by stray light An in-depth kinetic analysis of the single-turnover data was not possible under these experimental conditions Never-theless, the data enabled a comparison to be made
of the different B subtilis proteins as well as a comparison with the E coli protein
Figure 6 shows a single-turnover experiment with wild-type GTP cyclohydrolase II of B subtilis that was performed using an enzyme⁄ substrate ratio of 1 : 0.7 The reaction was characterized by a decrease in absor-bance at 252 nm and an apparently synchronous increase in absorbance at 292 nm The superposition
of spectra taken from the series showed an apparent isosbestic point at 278 nm, which suggests an apparent
0.160
0.120
0.080
0.040
0.0
240 260 280 300 320 340 360 380 400
Wavelength (nm)
0.1 0.51 10 50
Time (s) 5
Fig 6 Optical spectra from a stopped-flow experiment with
wild-type GTP cyclohydrolase II from Bacillus subtilis, using GTP as the
substrate.
Fig 7 Absorbance changes during single-turnover stopped-flow experiments with wild-type GTP cyclohydrolase II (A, B) and Construct E (C, D), using GTP as the substrate Reaction mixtures contained 50 m M Tris–HCl (pH 8.5), 100 m M NaCl, 10 m M MgCl2and 2 m M dithio-threitol The enzyme solution was mixed with substrate solution at a molar ratio of 1 : 0.7, at a temperature of 35 C.
Trang 6perfect reaction (data not shown) These findings are
all similar to the earlier findings made for GTP
cyclo-hydrolase II of E coli [13]
A comparison between the B subtilis wild-type
pro-tein and Construct E can be based on the progression
curves at selected wavelengths, as shown in Fig 7
Figure 7B,D also shows the total differentials of
absor-bance obtained at selected wavelengths versus time
The absorbance at 278 nm showed minimal variation
for both proteins
The wild-type and mutant proteins showed similar progression curves for the differentials at 310, 295 and
254 nm, suggesting that the two proteins under com-parison perform similarly under single-turnover condi-tions This unexpected finding will be discussed in more detail below
In similar experiments shown in Fig 8, the pro-tein⁄ substrate ratio was varied over a range of 1 : 0.7–
1 : 2.5 Progression curves are shown at 295 nm, a wavelength where the absorption is dominated by the nascent 2,5,6-triaminopyrimidinone motif present
in the hypothetical covalent intermediate 9 and the product 10, with only a minor contribution (by the substrate, GTP 1) to the absorbance Differentials of the absorbance at 295 nm (dA295⁄ dt) are shown in the frames on the right side of the Figure Whereas the curves for wild-type and mutant proteins were similar under conditions where there was a slight excess of protein over substrate, the similarity broke down under presteady-state conditions with an excess of
Fig 8 Numerical simulation of stopped-flow data of wild-type GTP cyclohydrolase II from Bacillus subtilis (A, B) and Construct E (C, D), using GTP as the substrate The enzyme solution was mixed with substrate solution at molar ratios of 1 : 0.7 (s), 1 : 1.3 ( ) and 1 : 2.5 ( ) Symbols represent the experimental data and lines represent the numerical simulation using the kinetic constants in Table 2 Data sets were analyzed using the program DYNAFIT [24].
Table 2 Single-turnover rate constants of different GTP
cyclo-hydrolase II proteins from Bacillus subtilis using GTP as the
substrate.
Trang 7substrate at the start of the reaction Under these
conditions, we observed a significantly higher initial
rate for the mutant protein compared with the
wild-type protein More specifically, dA295⁄ dt at t = 0 was
0.125 for the mutant protein and 0.085 for the
wild-type protein In the case of the wild-wild-type protein, a
plateau of dA295⁄ dt at a level of about 0.02, which
extended from about 10 to 30 s, followed the initial
steep decline By contrast, the reaction catalyzed by
the mutant protein was virtually complete within 30 s
As described in more detail below, this is best
explained by the hypothesis that the rate enhancement
observed for the mutant under steady-state conditions
is caused by differences in the rate of product release
Discussion
Four types of GTP cyclohydrolases are known to
cata-lyze the hydrolytic cleavage of the bond between C-8
and N-9 of the guanine moiety The ring-opening
reaction can be preceded and⁄ or followed by other
reaction steps catalyzed by the respective enzyme
Specifically, GTP cyclohydrolase I catalyzes the ring
opening of GTP, followed by hydrolytic
deformyla-tion, Amadori re-arrangement and ring closure
resulting in the production of dihydroneopterin
triphosphate, which serves as the first committed
precursor in the biosynthesis of tetrahydrofolate and
tetrahydrobiopterin (for review see Refs [17]) The
recently discovered MptA protein produces the
2¢,3¢-cyclophosphate of dihydroneopterin that is believed to
serve as a precursor for the biosynthesis of
tetrahydro-methanopterin, a one-carbon transfer cofactor of
methanogenic coenzymes [18] GTP cyclohydrolase II,
the subject of this article, is believed to catalyze the
release of phosphate from GTP, which is conducive to
the formation of a covalent guanylyl adduct that can
be resolved by a sequence of ring opening,
deformyla-tion and⁄ or phosphodiester cleavage resulting in the
production of 2, the first committed intermediate in
the biosynthesis of riboflavin The covalent adduct
remains to be confirmed by direct evidence, but the
recently reported 3D structure suggests that Arg128 is
the acceptor of the phosphodiester linkage in the
E coliprotein [19] GTP cyclohydrolase III of
Archae-bacteria catalyzes ring opening without accessory
reactions [20,21] The resulting
2-amino-5-formylami-no-6-ribosylamino-4(3H)-pyrimidinone
5¢-triphosphos-phate is believed to serve as the first committed
intermediate in the biosynthesis of riboflavin and of
the deazaflavin-type cofactor F420
Divalent cations appear to be essential for all known
GTP cyclohydrolases Specifically, the type I enzyme
uses a zinc ion that is coordinated by one cysteine resi-due and two histidine resiresi-dues The type II enzyme requires Mg2+ and a zinc ion that is coordinated by three cysteine residues It appears plausible that the opening of the imidazole ring involves a relatively large Gibbs free-energy barrier; however, the pre-steady-state analysis of the type I and type II enzymes indicates that the ring opening is not by any means the rate-determining step of the respective reaction trajec-tory; in the case of the E coli ortholog, which has been studied in some detail, the ring-opening reaction has a rate constant of 0.23 s)1 compared with a rate constant of 0.025 s)1for the overall reaction
Stopped-flow kinetic studies of wild-type and mutant GTP cyclohydrolase II of B subtilis, as described above, were conducted in close analogy to earlier studies on the E coli enzyme However, in con-trast to the E coli enzyme, the B subtilis proteins had
a marked tendency to form precipitates upon mixing with GTP This behavior suggests that substrate bind-ing is conducive to a more hydrophobic and aggrega-tion-prone state of the protein Owing to the resulting corruption of the optical readout by the stray light contribution, it was not possible to perform a detailed deconvolution of the stopped-flow kinetic data, in analogy to the earlier study performed with the E coli enzyme; despite this shortcoming, the data suggest that the kinetic profile of the B subtilis enzyme is indeed similar to that of the E coli enzyme, with the formation of the covalent adduct as a relatively slow initial step
Even without the opportunity to conduct a compre-hensive data deconvolution, stopped-flow analysis under single-turnover conditions, as well as presteady-state conditions, afforded useful information for comparison of the wild-type protein with the mutant Construct E Specifically, absorbance progression curves of the two proteins were similar under strict single-turnover conditions conducted with a molar excess of enzyme over substrate (Figs 7 and 8) By contrast, the kinetic differences became progressively larger when the substrate was proffered in excess (pre-steady-state conditions with a protein⁄ substrate ratio
up to a value of 1 : 2.5; Fig 8) Clearly, under these conditions, the mutant protein generated product at a higher overall rate than did the wild-type protein Moreover, these observations are perfectly in line with the steady-state analysis, indicating an approximately two-fold increased kcat of Construct E compared with the wild-type protein These findings are best explained by the hypothesis that the mutations in Construct E affect the rate of product release rather than the rate of product formation This hypothesis is
Trang 8also well in line with the increased Km values
deter-mined for Constructs C and E (Table 1); in fact,
preliminary data showed that various mutant proteins
selected for their increased Vmaxvalue also showed an
increase in their Kmvalue
The increased Km values of the mutant constructs
may be caused predominantly by an increased off-rate
for dissociation of the Michaelis complex For the time
being, we are limited to speculation because off-rates
have not been measured for any of the B subtilis
proteins under study Speculating further along these
lines, it is conceivable that an increased off-rate may
apply not only to the enzyme⁄ substrate complex
(Michaelis complex) but also to the enzyme⁄ product
complex That hypothesis is well in line with the
observed reaction acceleration under substrate
saturat-ing conditions An increased off-rate of the Michaelis
complex would be irrelevant for the catalytic rate
under saturating conditions
There is precedent for enzymes with substrate release
as the rate-limiting step for the overall reaction
Never-theless, it came as a surprise that the extensive
enzyme-evolution process conducted in this study
failed to increase the rate constant significantly for any
of the catalytical partial reactions sensus strictiori
(resulting in chemical modification of the reactant),
although the overall reaction velocity was increased
(via accelerated product release, as described earlier)
For the practical purpose of improving the
produc-tivity of a riboflavin-overproducing strain by
intro-ducing the improved GTP cyclohydrolase II domain
into a riboflavin-producing strain, it is irrelevant
whether the rate acceleration is caused by enhanced
substrate conversion or by enhanced product-release
rates The increase in Km accompanying the increase
in Vmax can be tolerated in the technical application
because the cellular GTP concentrations are well
above the Km, even for Construct E; moreover, this
enzyme would be working under substrate-saturating conditions in the in vivo situation
Based on the recently reported X-ray structure of the
E coli enzyme [22], the location of the mutations introduced by the enzyme-evolution strategy in relation
to its reaction center can be described at least approxi-mately In the crystal structure, the location of the cata-lytic site is clearly defined by the position of the zinc and magnesium ions and by bound GMP, one of the products of GTP cyclohydrolase II As shown in Fig 9, all mutations in Construct E are located outside the first amino acid shell of the substrate⁄ metal ion-binding cavity It appears quite plausible that remote mutations can be conducive to subtle deformations of the active-site cavity This could be conducive to a lowered ligand-binding affinity predominantly caused by an increased off-rate for both substrate and product An increased off-rate would not be conducive to the premature loss of intermediates because these are all covalently tethered to the protein
Experimental procedures
Materials 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5¢-triphosphate (10) was prepared as described previously [16] Protease inhibitors without EDTA, Taq polymerase, high-fidelity polymerase, restriction enzymes, DNase I, T4 ligase and the nucleotide mixture used for PCR were from Roche Diagnostics (Rotkreuz, Switzerland) Kanamycin sul-fate, ampicillin and most of the other fine-chemicals were supplied by Fluka (Buchs, Switzerland) LB (Luria–Bertani) medium was from Becton Dickinson (Basel, Switzerland)
Generation of mutant libraries For the generation of the ribA mutant library, the plasmid pQE60-ribANhis (Table 1) was used in which ribA from
B subtilis was cloned between the EcoR1 and the BamH1 sites of pQE60 (Table 1) The gene itself was slightly modi-fied by the addition of the DNA sequence motif 5¢-GAA TTCattaaagaggagaaattaact ATG AGA GGA TCT CAC CAT CAC CAT CAC CAT GGG ATC GAT CAT-3¢ in front of the start codon The modified ORF features an Nde1 site at the start codon and specifies a RibA protein carrying an N-terminal 6· His tag In order to introduce mutations exclusively into the cyclohydrolase II domain of ribA and not into the 3,4-dihydroxy-2-butanone (DHB) synthase domain, error-prone PCR was performed [(using the oligonucleotides ribA3S and ribA4AS (Table 4) as primers] only on the DNA fragment coding for the cyclo-hydrolase II domain Reaction mixtures for error-prone PCR contained 5 mm MgCl2, 0.7 mm MnCl2, 0.2 mm
Fig 9 Structure of wild-type GTP cyclohydrolase II from
Escheri-chia coli [22] The positions that are homologous to the mutations
in Construct E are marked in yellow.
Trang 9nucleotide triphosphates, 10 ng of template DNA, 2 lm of
each primer and 2.5 U of Taq polymerase in 50 lL of the
1· buffer supplied with the polymerase The reaction
condi-tions were as follows: step 1, 3 min, 95C; step 2, 30 s,
94C; step 3, 30 s, 52 C; step 4, 45 s, 72 C; and step 5,
7 min, 72C; steps 2 to 4 were repeated 35 times
For reconstruction of the entire ribA gene, the DHB
syn-thase domain was also amplified using the oligonucleotides
ribA1S and ribA2AS (Table 4) as primers under the
follow-ing conditions: 100 ng of template DNA, 2 lm of each
pri-mer, 2.5 U high-fidelity polymerase mixture and 0.2 mm
nucleotides in 50 lL of the 1· buffer supplied with the
poly-merase, using the heating protocol as described above Both
PCR products were purified by agarose-gel electrophoresis
and subsequent elution of the desired DNA fragments from
the gel In a third PCR, the purified PCR products were
assembled to create the complete ribA gene (100 ng of PCR
product 1, 100 ng of PCR product 2, 2.5 U high-fidelity
polymerase, 0.2 mm of nucleotides, 2 mm primer ribA1S
and 2 mm primer ribA4AS (Table 4) in 50 lL of the buffer
supplied with the polymerase; PCR protocol: step 1, 3 min,
95C; step 2, 30 s, 94 C, step 3, 30 s, 52 C; step 4, 2 min,
72C; and step 5, 7 min, 72 C; steps 2 to 4 were repeated
35 times) The PCR product was purified by using the PCR
purification kit from Qiagen The purified PCR product was
digested with EcoRI and BamHI and ligated into pQE60
(Table 3) also digested with EcoRI and BamHI The ligation
product was transformed into the riboflavin auxotrophic
strain E coli RB7 [15] [pREP4] (Table 3) Selection took
place on LB plates containing 100 mgÆmL)1 of ampicillin
Transformants were picked into 96-well plates containing
200 lL of LB medium (supplemented with 25 lgÆmL)1 of kanamycin and 100 lgÆmL)1of ampicillin) and were grown overnight Dimethylsulfoxide (15 lL per well) was added, and the plates were stored at)80 C For further rounds of mutagenesis, the original ribA gene was replaced with the improved mutants, as selected
Bacterial culture Aliquots (5 lL) from each well of a master plate were trans-ferred into a deep-well plate filled with 250 lL of LB medium (supplemented with 25 lgÆmL)1 of kanamycin and
100 lgÆmL)1 of ampicillin) per well The plates were incu-bated overnight (37C, 250 r.p.m.) on a rotary shaker The next morning, LB medium (1.2 mL) supplemented with
25 lgÆmL)1 of kanamycin and 100 lgÆmL)1 of ampicillin was added to each well The plates were incubated at 30C
on a rotary shaker at 250 r.p.m After 6 h, isopropyl
thio-b-d-galactoside was added to a final concentration of 0.5 mm The plates were incubated for another 16 h at 30C with shaking (250 r.p.m.) At the end of this incubation period, the cells were pelleted by centrifugation (20 min, 3220 g) and stored at)80 C
Screening Cell pellets in deep-well plates were suspended in 300 lL of
20 mm Tris–HCl (pH 7.5) containing 10 mm MgCl2, 15% sucrose, 0.1% Triton X-1000, 0.1 mgÆmL)1of lysozyme and
5 mgÆmL)1 of DNase I, and the recommended concentra-tion of Roche protease inhibitor without EDTA The plates were incubated at 20C under shaking at 200 r.p.m for
25 min, followed by centrifugation (20 min, 4000 g) Aliqu-ots (100 lL) of the supernatants were mixed with 150 lL of
a reaction mixture containing 100 mm Tris–HCl (pH 8.5),
10 mm MgCl2, 15 mm mercaptoethanol and 1.6 mm GTP The absorbance increase at 310 nm was monitored at 37C
In parallel experiments, 50-lL aliquots of the supernatants were mixed with 75 lL of a reaction mixture containing
50 mm Tris–HCl (pH 7.5), 10 mm MgCl2, 5 mm ribose-5-phosphate and 2.7 U of ribose 5-ribose-5-phosphate isomerase in a total volume of 100 lL The mixtures were incubated for
20 min at 37C A solution (100 lL) containing 2 m NaOH and 35 gÆL a-naphthol was added together with 50 lL of saturated creatine solution The mixture was incubated at
20C for periods of 60 to 120 min, and the absorbance at
525 nm was determined [23]
Protein purification Frozen E coli cell mass (25 g) was thawed in 60 mL of
50 mm Tris–HCl (pH 8.0), containing 0.3 m sodium chlo-ride and 10 mm magnesium chlochlo-ride The cells were
Table 3 Microorganisms and plasmids used in this study.
Strain or plasmid
Genotype or relevant characteristics
Reference
or source Escherichia coli Rib7 thi leu pro lac ara xyl
endA recA hsd r - m
-pheS supE44 rib
[15]
Plasmids
expressing lacI
Quiagen Inc.
E coli
Quiagen Inc.
pQE60-ribA-Nhis pQE60 with an N-terminally
tagged ribA from Bacillus subtilis
This study
Table 4 Oligonucleotides used in this study.
Oligonucleotide Nucleotide sequence (5¢- to 3¢)
Trang 10disrupted by ultrasonic treatment, and the suspension was
centrifuged The supernatant was applied to a column of
Ni-chelating Sepharose FF (GE Healthcare Europe GmbH,
Otelfingen, Switzerland; column volume 20 mL), which had
been equilibrated with 50 mm Tris–HCl (pH 8.0) containing
0.3 m sodium chloride and 10 mm magnesium chloride
(flow rate, 2 mLÆmin)1) The column was washed with
100 mL of the equilibration buffer and was then developed
with a gradient of 0–200 mm imidazole in 50 mm Tris–HCl
(pH 8.0) containing 0.3 m sodium chloride, 10 mm
magne-sium chloride and 5% glycerol (total volume, 100 mL)
Fractions were combined and dialyzed overnight against
50 mm Tris–HCl (pH 8.5) containing 100 mm sodium
chlo-ride, 10 mm magnesium chlochlo-ride, 2 mm dithiothreitol and
5% glycerol The enzyme was stored at 4C
Steady-state kinetics
Reaction mixtures contained 50 mm Tris–HCl (pH 8.5),
100 mm NaCl, 10 mm MgCl2, 2 mm dithiothreitol and
pro-tein in a total volume of 400 lL Experiments were
per-formed at 30C The reaction was initiated by the addition
of GTP to a predetermined concentration (0.017–1.7 mm)
The assay was monitored photometrically at 310 nm
Reac-tion rates were calculated using an absorpReac-tion coefficient of
7.43 mm)1Æcm)1 for
2,5-diamino-6-ribosylamino-4(3H)-pyri-midinone 5¢-phosphate
Stopped-flow kinetic experiments
Experiments were performed using an SFM4⁄ QS apparatus
from Bio-Logic (Claix, France) equipped with a linear
array of three mixers and four independent syringes The
content of a 1.5-mm light path quartz cuvette behind the
last mixer was monitored using a Tidas diode array
spec-trophotometer (200–610 nm) equipped with a 15 W
deute-rium lamp as the light source (J&M Analytische Meß- und
Regeltechnik, Aalen, Germany) The reaction buffer
con-tained 50 mm Tris–HCl (pH 8.5), 100 mm NaCl, 10 mm
MgCl2 and 2 mm dithiothreitol The enzyme solution was
mixed with substrate solution at a temperature of 35C
and a total flow rate of 4 mLÆs)1 The calculated dead time
was 7.6 ms Spectra integrated over 96 ms were recorded at
intervals of 100 ms
References
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