The crystal structure of coenzyme B12-dependent glycerol dehydratasein complex with cobalamin and propane-1,2-diol Mamoru Yamanishi1, Michio Yunoki1, Takamasa Tobimatsu1, Hideaki Sato1,
Trang 1The crystal structure of coenzyme B12-dependent glycerol dehydratase
in complex with cobalamin and propane-1,2-diol
Mamoru Yamanishi1, Michio Yunoki1, Takamasa Tobimatsu1, Hideaki Sato1, Junko Matsui1, Ayako Dokiya1, Yasuhiro Iuchi1, Kazunori Oe1, Kyoko Suto2, Naoki Shibata2, Yukio Morimoto2, Noritake Yasuoka2 and Tetsuo Toraya1
1
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama;2Department of Life Science, Himeji Institute of Technology, Hyogo, Japan
Recombinant glycerol dehydratase of Klebsiella pneumoniae
was purified to homogeneity The subunit composition of
the enzyme was most probably a2b2c2 When (R)- and
(S)-propane-1,2-diols were used independently as substrates, the
rate with the (R)-enantiomer was 2.5 times faster than that
with the (S)-isomer In contrast to diol dehydratase,an
iso-functional enzyme,the affinity of the enzyme for the
(S)-isomer was essentially the same or only slightly higher than
that for the (R)-isomer (Km(R)/Km(S)¼ 1.5) The crystal
structure of glycerol dehydratase in complex with
cyanoco-balamin and propane-1,2-diol was determined at 2.1 A˚
resolution The enzyme exists as a dimer of the abc
hetero-trimer Cobalamin is bound at the interface between the a
and b subunits in the so-called base-on mode with
5,6-dimethylbenzimidazole of the nucleotide moiety
coordina-ting to the cobalt atom The electron density of the cyano
group was almost unobservable,suggesting that the
cyano-cobalamin was reduced to cob(II)alamin by X-ray
irradi-ation The active site is in a (b/a)8barrel that was formed by a
central region of the a subunit The substrate propane-1,2-diol and essential cofactor K+are bound inside the (b/a)8
barrel above the corrin ring of cobalamin K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues These structural features are quite similar to those of diol dehydratase A closer contact between the a and b subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydra-tase Although racemic propane-1,2-diol was used for cry-stallization,the substrate bound to glycerol dehydratase was assigned to the (R)-isomer This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by gly-cerol
Keywords: coenzyme B12; adenosylcobalamin; glycerol dehydratase; crystal structure; radical enzyme catalysis
Adenosylcobalamin is one of the most unique compounds
in nature It is a water-soluble organometallic compound
possessing a Co–C r bond and serves as a cofactor for
enzymatic radical reactions including carbon skeleton
rearrangements,heteroatom eliminations and
intramolecu-lar amino group migrations [1] Diol dehydratase (EC
4.2.1.28) of Klebsiella oxytoca is an adenosylcobalamin
(AdoCbl1) dependent enzyme that catalyzes the conversions
of 1,2-diols,such as propane-1,2-diol,glycerol,and 1,
2-ethanediol,to the corresponding aldehydes [2,3] (Fig 1)
This enzyme has been studied intensively to establish the
mechanism of action of AdoCbl [4–7] The structure–
function relationship of the coenzyme has also been
investigated extensively with this enzyme [5–8] Recently,
we have reported the three-dimensional structures of its
complexes with cyanocobalamin [9] and adeninylpentylco-balamin [10] and theoretical calculations of the entire energy profile along the reaction pathway with a simplified model [11–13] In this sense,together with methylmalonyl-CoA mutase [14],glutamate mutase [15],and class II ribonucleo-tide reductase [16],diol dehydratase,is one of the most suitable systems with which to study the structure-based mechanisms of the AdoCbl-dependent enzymes [17,18] Glycerol dehydratase (EC 4.2.1.30) catalyzes the same reaction (Fig 1) as diol dehydratase [19–21] Although this enzyme is isofunctional with diol dehydratase,these two enzymes bear different physiological roles in the bacterial metabolisms [6,7] Selected genera of Enterobacteriaceae, such as Klebsiella and Citrobacter,produce both glycerol and diol dehydratases,but the genes for them are inde-pendently regulated [22–25]: glycerol dehydratase is induced when Klebsiella pneumoniae grows in the glycerol medium, whereas diol dehydratase is fully induced when it grows in the propane-1,2-diol-containing medium, but only slightly
in the glycerol medium Glycerol dehydratase is a key enzyme for the dihydroxyacetone (DHA) pathway [23,26,27], and its genes are located in the DHA regulon [28,29] On the other hand, diol dehydratase is a key enzyme for the anaerobic degradation of 1,2-diols [30,31], and its genes are located in the pdu operon [32–34] Furthermore, although glycerol and diol enzymes are similar in their
Correspondence to T Toraya,Department of Bioscience and
Biotechnology,Faculty of Engineering,Okayama University,
Tsushima-naka,Okayama 700–8530,Japan.
Fax: + 81 86 2518264,E-mail: toraya@biotech.okayama-u.ac.jp
Abbreviations: AdoCbl,adenosylcobalamin; a D , b D ,and c D , a, b,and
c subunits of diol dehydratase; a G , b G ,and c G , a, b,and c subunits of
glycerol dehydratase; buffer A,0.05 M potassium phosphate buffer
(pH 8); IPTG,isopropyl thio-b- D -galactoside.
(Received 11 June 2002,revised 23 July 2002,accepted 25 July 2002)
Trang 2subunit structures,there are several distinct differences
between them in the following properties: the rate of suicide
inactivation by glycerol,substrate spectrum,monovalent
cation requirement,affinity for cobalamins,and
immuno-chemical cross-reactivity [6,7]
In this paper,we report the method of purifying
recombinant apoglycerol dehydratase from overexpressing
Escherichia colicells and the crystal structure of glycerol
dehydratase in complex with cyanocobalamin and
propane-1,2-diol We intended to explain the above-mentioned
differences between two dehydratases by comparing the
three-dimensional structure of this enzyme with that of diol
dehydratase [9,10]
E X P E R I M E N T A L P R O C E D U R E S
Materials
Crystalline AdoCbl was a gift from Eizai,Tokyo,Japan
DEAE-cellulose was purchased from Wako,Osaka,Japan
The other chemicals were analytical grade reagents
Preparation of expression plasmids for His6-tagged
glycerol dehydratase and its His6-tagged b subunit
DNA segments encoding carboxyl terminal region of the
glycerol dehydratase a subunit was amplified by PCR using
pUSI2E(GD) [28], pfu DNA polymerase (Stratagene) and
pairs of primers 5¢-TCTGAGTGCGGTGGAAGAGATG
ATGAAGCG-3¢ and 5¢-AGATCTTATTCAATGGTGT
CGGGCTGAACC-3¢ and digested with EcoRV and BglII
Resulting 210-bp fragment was ligated with the 1.5-kb
HindIII-EcoRV fragment from pUSI2E(GD) and pUSI2E
digested with HindIII and BglII to yield pUSI2E(aG) DNA
segments encoding the b and c subunits of glycerol
dehydratase were amplified by PCR using pairs of primers
5¢-CATATGCAACAGACAACCCAAATTCAGCCC-3¢
and 5¢-AGATCTTATCACTCCCTTACTAAGTCGATG-3¢
for the b subunit and 5¢-CATATGAGCGAGAAAACCA
TGCGCGTGCAG-3¢ and 5¢-AGATCTTAGCTTCCTTT
ACGCAGCTTATGC-3¢ for the c subunit The segments
were digested with NdeI and BglII and ligated with 3.5-kb
ApaI-BglII fragment and 1.5-kb ApaI-NdeI fragment from
pUSI2E(bD) [35] to yield pUSI2E(bG) and pUSI2E(cG),
respectively Plasmid pUSI2E(bG) was digested with NdeI
and BglII Resulting 600-bp NdeI-BglII fragment was
inserted into the NdeI-BglII region of pET19b to produce
pET19b(H6bG) A pair of synthetic oligonucleotides,
5¢-TATGGGCAGCAGCCATCATCATCATCATCACA
GCAGCGGCCTGGTGCCGCGCGGCAGCAC-3¢ and
5¢-TAGTGCTGCCGCGCGGCACCAGGCCGCTGCT
GTGATGATGATGATGATGGCTGCTGCCCA-3¢
were hybridized and inserted to the NdeI site of pUSI2E(cG)
to produce pUSI2E(H6Gc) pUSI2E(bG) was digested with
BamHI and BglII The resulting 0.7-kb DNA fragment was
ligated with BglII-digested pUSI2E(aG) to produce pUSI2E(aGbG) The 0.5-kb BamHI–BglII fragment from pUSI2E(H6cG) was ligated with BglII-digested pUSI2E(H6cG) to produce plasmid pUSI2E(aGbGH6cG) Purification of recombinant glycerol dehydratase Glycerol dehydratase was purified from recombinant Escherichia coli by a conventional procedure (method 1)
or Ni-nitrilotriacetate affinity chromatography (method 2) Substrate propane-1,2-diol was added to all the buffers used throughout the purification steps to minimize dissociation
of the enzyme into components A and B [36] All operations were carried out at 0–4C
Method 1 Recombinant E coli JM109 harboring expres-sion plasmid pUSI2E(GD) [28] was aerobically grown at
37C in Luria–Burtani (LB) medium containing propane-1,2-diol (0.1%) and ampicillin (50 lgÆmL)1) to D600 0.9, induced with 1 mMisopropyl thio-b-D-galactoside (IPTG) for 5 h,and harvested by centrifugation Harvested cells were resuspended in buffer A (0.05Mpotassium phosphate buffer; pH 8) containing 2 mM phenylmethanesulfonyl fluoride and disrupted by sonication for 10 min,followed
by centrifugation Ammonium sulfate was added to the supernatant to a final concentration of 50% saturation After 60 min at 4C,the suspension was centrifuged,and the precipitate was re-dissolved in buffer A The solution was subjected to gel filtration on Sepharose 6B,and peak fractions containing the enzyme were pooled,dialyzed for
12 h against 40 volumes of 1.5 mM potassium phosphate buffer (pH 8) containing 0.2% propane-1,2-diol with a buffer exchange,and loaded on to a hydroxyapatite column which had previously been equilibrated with 2 mM potas-sium phosphate buffer (pH 8) containing 0.2% propane-1,2-diol After washing the column with 5 mM potassium phosphate buffer (pH 8) containing 0.2% propane-1,2-diol, the enzyme was eluted with 13 mM potassium phosphate buffer (pH 8) containing 2% propane-1,2-diol The eluate was concentrated and loaded on to a Sephadex G-200 column which had previously been equilibrated with 20 mM
potassium phosphate buffer (pH 8) containing 2% pro-pane-1,2-diol The enzyme was eluted with the same buffer, and peak fractions containing the enzyme were pooled Method 2 Recombinant E coli JM109 harboring pUSI2E(aGbGH6cG) was aerobically grown at 30C in terrific broth containing propane-1,2-diol (0.1%) and ampi-cillin (50 mg mL)1) to D600 0.9,induced with 1 mM
IPTG for 7 h,and harvested by centrifugation Harvested cells were resuspended in buffer A containing 2 mM
phenylmethanesulfonyl fluoride and sonicated as described above The extract containing His6-tagged enzyme was mixed with an equal volume of buffer A containing 20 mM
imidazole and 600 m KCl and loaded on to an
Fig 1 Conversion of 1,2-diols to the corresponding aldehydes by diol dehydratase.
Trang 3Ni-nitrilotriacetate agarose gel (Qiagen GmbH,Germany)
column which had previously been equilibrated with buffer
A containing 10 mM imidazole and 300 mM KCl After
washing the column with buffer A containing 10 mM
imidazole and 300 mM KCl, the enzyme was eluted with
buffer A containing 50–100 mM imidazole and 300 mM
KCl After dialysis against 40 volumes of 50 mMTrisHCl
buffer (pH 8) containing 2% propane-1,2-diol,150 mM
KCl and 2.5 mMCaCl2,His6-tagged enzyme was digested
with thrombin at 25C for 120 min and run through the
Ni-nitrilotriacetate agarose column to remove the His6-tag
peptide Because a part of the enzyme had lost the b subunit,
the enzyme solution was concentrated and supplied with
purified b subunit by incubation at 30C for 30 min,
followed by Sepharose 6B gel filtration to remove
unbound,excess b subunit The b subunit was purified from
E coliBL21 (DE3) carrying pET19b(H6bG),as described
above for glycerol dehydratase
Enzyme and protein assays
Glycerol dehydratase activity was determined by a
3-methyl-2-benzothiazolinone hydrazone method [37] or
an NADH–alcohol dehydrogenase coupled method [38] at
37C Propane-1,2-diol was used as a substrate for routine
assays because glycerol acts as both a good substrate and a
potent suicide inactivator [39] One unit of glycerol
dehy-dratase is defined as the amount of enzyme activity that
catalyzes the formation of 1 lmol of propionaldehyde per
minute under the assay conditions Protein concentration of
crude enzyme was determined by the method of Lowry et al
[40] with crystalline bovine serum albumin as a standard
The concentration of purified enzyme was determined by
measuring the absorbance at 280 nm The molar absorption
coefficient at 280 nm calculated by the method of Gill and
von Hippel [41] for this enzyme is 112 100M )1cm)1
Separation of the enzyme into components A and B
A purified preparation of the enzyme (80 units) was applied
to a column (bed volume,2.0 mL) of DEAE cellulose that
had been equilibrated with 10 mM potassium phosphate
buffer (pH 8) containing 10 mM propane-1,2-diol After
washing the column with 50 mL of 10 mM potassium
phosphate buffer (pH 8),components A and B were eluted
successively with 5 mL of 10 mM potassium phosphate
buffer (pH 8) containing 40 mMKCl and then with 50 mL
of 10 mM potassium phosphate buffer (pH 8) containing
300 mM KCl,respectively Five-milliliter fractions were
collected Neither component alone was active,while the
enzyme activity was restored upon addition of the other
component Therefore,components A and B were assayed
by adding an excessive amount of one component and
making the other rate-limiting
PAGE and activity staining of glycerol dehydratase
PAGE was performed under nondenaturing conditions as
described by Davis [42] in the presence of 0.1M
propane-1,2-diol [32],or under denaturing conditions as described by
Laemmli [43] Protein was stained with Coomassie brilliant
blue G-250 Densitometry was carried out by Personal
Scanning Imager PD110 (Molecular Dynamics) Activity
staining for glycerol dehydratase was performed as des-cribed previously for diol dehydratase [32] The apparent molecular weight of the enzyme was estimated by the nondenaturing PAGE on a Multigel 2–15% gradient gel (Daiichi Pure Chemicals,Tokyo,Japan) [44]
Kinetic analysis of the enzyme Substrate-free enzyme used for measuring Km values for substrates was obtained by gel filtration on Sephadex G-25 or dialysis for 3 days against 500 volumes of buffer
A with several changes One-minute assay was employed for measurement of Kmfor glycerol,as glycerol induces suicide inactivation of the enzyme [39] Apparent Kmvalues for substrates and AdoCbl were determined at an AdoCbl concentration of 15 lM and at a fixed propane-1,2-diol concentration of 100 mM,respectively
EPR measurements The complex of glycerol dehydratase with adenosylcobin-amide 3-imidazolylpropyl phosphate [45] was formed by incubating apoenzyme (100 units,4.55 nmol) at 25 C for
5 min with 50 nmol of the coenzyme analog in 0.65 mL of buffer A (pH 8) under a nitrogen atmosphere Propane-1,2-diol (50 lmol) was then added After the mixture had been incubated at 25C for an additional 30 min,the mixture was quickly frozen in an isopentane bath (cooled to
)160 C) and then in a liquid nitrogen bath The sample was transferred to an EPR cavity and kept at)130 C with
a cold nitrogen gas flow controlled by a JEOL JES-VT3A temperature controller EPR spectra were taken at)130 C
on JEOL JES-RE3X spectrometer modified with a Gunn diode X-band microwave unit under the same conditions as those described for diol dehydratase [46]
Crystallization and data collection Purified glycerol dehydratase (64 mgÆmL)1) in 20 mM
potassium phosphate buffer (pH 8) containing 2% pro-pane-1,2-diol was converted to the enzymeÆcyanocobal-aminÆpropane-1,2-diol complex by the same method as that for diol dehydratase [9] except that lauryldimethylamine oxide was not included The complex was crystallized by the sandwich-drop vapor diffusion method at 4C X-ray diffraction data were collected at 100 K using the Quantum-4R CCD detector (ADSC) on the BL40B2 beam line at SPring-8,Japan (Table 1) Reflection data were indexed, integrated and scaled using the programs Mosflm and SCALA in the CCP4 suite [47] with DPS [48]
Structure determination and refinement The structure of the enzyme was determined and refined using the programCNS[49] The models were built using Xfit of XTALVIEW [50] and checked by PROCHECK No noncrystallographic symmetry (NCS) restraints were enforced during whole refinements For adjusting the positions of atoms,a composite-omit map (2Fo) Fc) and
an Fo) Fcmap were used
A data set obtained was up to 2.0 A˚ resolution Crystallographic data are listed in Table 1 The a, b and c subunits of glycerol dehydratase show substantially high
Trang 4homology to the corresponding subunits of diol
dehydra-tase: their identities are 71,58 and 54%,respectively,and
their similarities 87,78 and 73%,respectively [28] We
started the structure determination by the molecular
replacement method with the abc heterotrimer unit of diol
dehydratase from Protein Data Bank (PDB) entry 1DIO [9]
as a reference structure After a cross-rotation search,
multiple translation searches were performed,and the
monitor and the packing values were checked to determine
the result from the candidates We concluded that there
is an (abc)2dimer in an asymmetric unit of the cell The
calculated VMvalue was 3.03 A˚3ÆDa)1 (VM¼ Vcell/ZÆMr,
where Vcelland Z are the unit cell volume and the number of
protein molecules per unit cell,respectively)
At this stage,the residues of diol dehydratase were
replaced with the corresponding residues of glycerol
dehydratase After one set of rigid-body refinement and
simulated annealing were applied,a composite-omit map (2Fo) Fc) was calculated On this map,distinct electron densities were observed in the positions next to N- and C-ends of each chain They could be assigned to certain amino-acid residues,because the C-terminal three residues
of aD,the N-terminal 45 residues of bD,and the N-terminal
36 residues and the C-terminal three residues of cDwere missing in the reported structure of diol dehydratase [9] In addition, aGand bGare longer by one amino acid than aD
in the N-terminal and by three than bDin the C-terminal, respectively In the final structure,all residues of the a chain (Met1–Glu555),all but N-terminal 10 residues of the b chain (Phe11–Glu194),and all but N-terminal 3 residues of the c chain (Lys4–Ser141) could be assigned to the electron density map In glycerol dehydratase,the numbers of missing residues were smaller than those of diol dehydra-tase We have not determined yet whether these missing residues are actually truncated by hydrolysis or not visible because of their high mobility
After the successive repeats of modeling,energy-minimi-zation and simulated annealing,about 900 water molecules were picked up,and B-factors for all the atoms were refined The structure showed good stereochemistry with root-mean-square (rms) deviations of 0.006 A˚ from the ideal bond length and 1.30 from ideal bond angles The resulting
Rwork and Rfreewere 0.208 and 0.248,respectively,in the resolution range of 45.0–2.1 A˚
Unless otherwise stated,structural figures were created withMOLSCRIPT[51] andRASTER3D[52]
Accession number The atomic coordinates have been deposited in the Protein Data Bank with an accession code of 1IWP
R E S U L T S A N D D I S C U S S I O N Purification and characterization of recombinant glycerol dehydratase
Recombinant nontagged glycerol dehydratase was purified
by a conventional method As shown in Table 2,glycerol dehydratase overexpressed in E coli was purified by ammonium sulfate fractionation and chromatography on Sepharose 6B,hydroxyapatite,and Sephadex G-200
Table 2 Purification of recombinant glycerol dehydratase.
Purification step
Total activity (units)
Total protein (mg)
Specific activity (UÆmg)1)
Yield (%)
Purification (fold) Method 1a
Method 2 b
a Purification from 4.7 g of wet cells b Purification from 13 g of wet cells.
Table 1 Statistics of data collection and structure determination The
values in parentheses are for the highest resolution shell.
Unit cell (A˚)
Measured reflections 958 394 Unique reflections 130 635 Completeness (%) 99.6 (97.6)
R merge 0.097 (0.38) Refinement Resolution range (A˚) 45.0–2.1
Completeness (%) 99.9
R work a
0.208
a
R-factor=S||F o | ) |F c ||/S|F o | R work or the working R-factor is
calculated on the 90% of the observed reflections used for the
refinement b R free or the free R-factor is calculated on the 10% of
reflections excluded from the refinement.
Trang 5(method 1) The enzyme was purified 4.3-fold in a yield of
63% Specific activity was about 65 units/mg SDS/PAGE
analysis showed that three bands with an Mrof 61 000 (a),
22 000 (b) and 16 000 (c) (marked with an arrowhead) were
overexpressed in E coli carrying pUSI2E(GD) (Fig 2A)
and progressively enriched upon purification,and that only
these subunits were found in the purified preparation of the
enzyme When the enzyme was electrophoresed under
nondenaturing conditions in the presence of substrate (Fig 2B),however,two bands were seen upon protein stain-ing The ratio of the upper protein band to the lower one was estimated to be approximately 2 by densitometric scanning Activity staining of the enzyme indicated that the upper band reconstituted catalytically active holoenzyme with added AdoCbl,but the lower one did not (data not shown) The mobility of the upper band was identical with that of active glycerol dehydratase in the extract of K pneumoniae ATCC
25955 (data not shown) Two-dimensional PAGE showed that the upper band consisted of the a, b and c subunits in a molar ratio of 1.0 : 1.0 : 1.2 The lower band was composed
of the a and c subunits in a molar ratio of 1.0 : 0.9 When the purified enzyme was subjected to nondenaturing PAGE on a Multigel 2/15 gradient gel [44],two bands appeared upon protein staining,and only the upper one stained upon activity staining (data not shown) The Mrvalues for the upper and lower bands were 220 000 and 200 000,respectively These data suggest that the subunit compositions of the proteins in the upper and lower bands are most likely a2b2c2 (active apoenzyme,predicted molecular mass of 196 236 Da) and
a2c2(component B,predicted molecular mass of 153 526 Da),respectively
In order to confirm this assignment for the lower band,
we attempted to see what happens if component A is added
to the purified preparation of the enzyme We prepared components A and B by separation of purified enzyme upon DEAE–cellulose chromatography in the absence of substrate Recoveries of activity of components A and B were 24% and 10%,respectively,although weak glycerol dehydratase activity was observed in the component B fraction alone SDS/PAGE analysis showed that compo-nents A and B contain the b subunit alone and a 1 : 1 mixture of the a and c subunits,respectively (Fig 2C) Thus,it was concluded that the inactive protein contam-inated in the purified enzyme (lower band in Fig 2B) is component B When an excessive amount of component A was added to the purified enzyme,propane-1,2-diol-dehy-drating activity increased by 59% PAGE analysis under nondenaturing conditions showed that the catalytically inactive lower band seen in the purified enzyme was converted to the active upper band upon the addition of component A (Fig 2D) Three bands were observed in the
component B fraction upon nondenaturing PAGE Posi-tions of the top and middle bands coincided well with the two bands observed with the purified enzyme Thus,it was suggested that the middle and top minor bands of the
component B fraction correspond to component B (a2c2) and a trace of contaminating active apoenzyme, a2b2c2 The bottom major band that had newly appeared has not been identified yet
For brevity,we developed a quick purification method (method 2) for His6-tagged component A and glycerol dehydratase His6-tagged enzyme was overexpressed in
E coli and purified with an Ni-affinity column After removal of His6 tag by digestion with thrombin,followed
by passage through the Ni-nitrilotriacetate column,the enzyme obtained was incubated with excess component A and run through a Sepharose 6B column Highest specific activity of the enzyme (120 UÆmg)1) was obtained by this simple procedure (Table 2) PAGE analysis under denatur-ing (Fig 2E) and nondenaturdenatur-ing conditions (Fig 2F) indicated that the purified enzyme was contaminated with
Fig 2 Characterization of purified glycerol dehydratase by PAGE.
SDS/PAGE (A,E) and nondenaturing PAGE (B,F) analyses of the
enzyme at each purification step of method 1 (A,13.5% gel; B,7.5%
gel) and method 2 (E,12% gel; F,7.5% gel) Resolved components A
and B were also subjected to SDS/PAGE (C) and nondenaturing
PAGE (D) Proteins were stained by Coomassie brilliant blue G.
Molecular mass markers,Sigma SDS-7 L Positions of the a, b and c
subunits and their complexes are indicated with arrowheads in the
right H 6 -a 2 b 2 c 2 and H 6 -c represent the His6-tagged a 2 b 2 c 2 complex
and His6-tagged c subunit,respectively.
Trang 6component B,and that the contaminating component B
recombined with the b subunit (component A) to form
a2b2c2 that resisted dissociation upon Sepharose 6B
column chromatography As a result,the enzyme was
purified 5.0-fold in a yield of 18% This method was
employed for crystallization of glycerol dehydratase
Kinetic parameters and stereospecificity
of recombinant glycerol dehydratase
Kinetic constants of the purified recombinant glycerol
dehydratase for AdoCbl,propane-1,2-diol,and glycerol
were in reasonable agreement with those reported previously
for the enzyme from K pneumoniae (Table 3),suggesting
that the recombinant enzyme and the enzyme from
K pneumoniaeare not distinguishable When (R)- and
(S)-propane-1,2-diols were used independently as substrates, the
rate with the (R)-enantiomer was 2.5 times faster than that
with the (S)-isomer (Table 3) The affinity of the enzyme for
the (S)-isomer was essentially the same or only slightly
higher than that for the (R)-isomer [Km(R)/Km(S)¼ 1.5] This
preference to the (S)-isomer is significantly less marked than
that reported with diol dehydratase [53,54]
EPR spectroscopic evidence for the ‘base-on’ mode
of cobalamin binding
To identify the Co-coordinating base,EPR spectra of the
suicidally inactivated complexes of the enzyme with
unla-beled and [imidazole-15N2]-labeled adenosylcobinamide
3-imidazolylpropyl phosphate were compared The EPR
spectra obtained with these analogs were exactly the same as
those reported for diol dehydratase [46,55] (data not
shown) With the unlabeled imidazolyl analog,each line
of the hyperfine octet (coupling constant,10.6 mT) showed
superfine splitting into triplets (coupling constant,1.9 mT)
With the [imidazole-15N2]-labeled analog,on the other
hand,the hyperfine lines (coupling constant,10.7 mT)
showed superhyperfine splitting into doublets (coupling
constant,2.7 mT) The ratio of the coupling constant with
14N (A14N) to that with15N (A15N) was 0.704,which is in
good agreement with the theoretical one that can be
calculated as follows:
A14N=A15N ¼ c14N= 15N ¼ 0:713 ðtheoreticalÞ
where c is a gyromagnetic ratio These lines of evidence
indicated that the axial ligand to Co(II) is the imidazole of
the coenzyme analog Therefore,it is evident that,like diol
dehydratase [46,56], glycerol dehydratase binds AdoCbl in
the so-called base-on mode This conclusion is consistent
with the finding of Poppe et al that p-cresolylcobamide coenzyme is inactive and serves as an inhibitor for diol and glycerol dehydratases [57]
Overall structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol
The crystal structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol was determined at 2.1 A˚ resolution by the molecular replacement method The schematic view of the overall structure is shown in Fig 3A The enzyme exists as a dimer of the abc heterotrimer There
is a noncrystallographic twofold axis around the center of Fig 3A The structure of an abc heterotrimer unit is shown
in Fig 3B The central region of the a subunit constitutes the (b/a)8 barrel,the so-called TIM (triosephosphate isomerase) barrel Propane-1,2-diol, a substrate, and K+,
an essential cofactor,are bound inside the barrel The active site-cavity is covered by the corrin ring of cobalamin that is bound on the interface of the a and b subunits Two a subunits form dimer a2to which two b and two c subunits are bound separately This structure is quite similar to that
of diol dehydratase [9] To compare the Catrace between glycerol and diol dehydratases,the abc structure of glycerol dehydratase superimposed on the structure of diol dehy-dratase is shown in Fig 3C with the rms deviation ranges differently colored It is clear that deviations of atoms in the
b and c subunits are relatively large,although the rms deviation of Ca atoms in the a subunit was less than 1.0 A˚ The Kmvalues of glycerol dehydratase for AdoCbl is 40–100 times lower than that of diol dehydratase (Table 3) Such higher affinity of glycerol dehydratase for AdoCbl may be explained by the closer contact between the a and b subunits
in which cobalamin sits
Glycerol dehydratase is isofunctional with diol dehydra-tase,and its amino acid sequences of the a, b and c subunits are 71,58 and 54% identical with those of diol dehydratase [28] They are immunologically different or only slightly cross-reactive under nondenaturing conditions [22],but anti-(K oxytoca diol dehydratase) antiserum cross-reacted with K pneumoniae glycerol dehydratase to some extent under denaturing conditions (data not shown) As shown in Fig 3D,most of the amino acid residues that are not conserved between these enzymes are located on the surface
of the glycerol dehydratase molecule,whereas the conserved residues constitute the core part of the enzyme This fact explains the above-mentioned very low cross-reactivity of glycerol dehydratase with anti-(diol dehydratase) antiserum under nondenaturing conditions and its low but distinct cross-reactivity under denaturing conditions
Table 3 Kinetic constants for the coenzyme and substrates.
Glycerol dehydratase
K m for AdoCbl (n M )
K m (k cat ) [m M (s)1)]
Glycerol Propane-1,2-diol (R)-Propane-1,2-diol (S)-Propane-1,2-diol
(604 ± 32) d
0.27 ± 0.10 (244 ± 23) d
a
From [39].bFrom [61].cFrom [62].dMean ± SD, n ¼ 11–14.
Trang 7Cobalamin-binding site and the conformation
of bound cobalamin
Figure 4A depicts the structure of the active site in the (b/a)8
barrel Substrate propane-1,2-diol and K+are locked in the
active-site cavity that is isolated from a bulk of water by the
corrin ring of cobalamin Figure 4B shows the structure
around the enzyme-bound cobalamin The cobalamin
molecule is bound between the a and b subunits in the
so-called base-on mode) that is,with the
5,6-dimethyl-benzimidazole moiety coordinating to the cobalt atom
Again,this binding mode is quite similar to that of diol
dehydratase [9] Crystallographic indication of the base-on
mode of cobalamin binding in class II ribonucleotide
reductase of Lactobacillus leichmannii has also been
repor-ted very recently by Drennan and coworkers [16] The
amino acid residues of diol dehydratase that are
hydrogen-bonded to the peripheral amide side chains of the corrin ring
[9] are all conserved in glycerol dehydratase as well In addition to these conserved residues,the hydroxyl group of Serb122 is hydrogen-bonded to the amide oxygen of the g-acetamide side chain of the corrin ring in glycerol dehydratase (red dotted line in Fig 4B) In diol dehydra-tase,the corresponding residue is Prob155 that cannot form the hydrogen bond Furthermore,the lengths of the hydrogen bonds are shorter with five amino acid residues and longer with four residues in glycerol dehydratase than those in the diol enzyme These may be reminiscent of the fact that the former enzyme binds AdoCbl much more tightly than the latter enzyme (Table 3)
In the case of glycerol dehydratase,no electron density
of the cyanide ligand was seen even though diffraction data were collected at 100 K The Co–N bond distance between the cobalt atom and N(3) of 5,6-dimethylbenzimidazole in the glycerol dehydratase-bound cobalamin is 2.48 A˚ This value is close to that in the diol dehydrataseÆcobalamin
Fig 3 Structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol (A) Overall structure One abc heterotrimer unit of the two was drawn in a schematic model,and the other in a tube model a, b and c chains are colored in blue,yellow and orange,respectively,darkening continuously from the N- to C-terminal sides Eight b strands constituting the (b/a) 8 barrel are drawn in cartoon Cobalamin,propane-1,2-diol and
K + are shown as CPK models colored in pink,green,and cyan,respectively (B) Structure of the abc heterotrimer unit (C) Stereoview of the C a
traces of the abc heterotrimer unit The corresponding traces of diol dehydratase are drawn in gray Root mean square deviation (A˚) from the diol dehydratase structure: dark blue,< 0.5; light blue,0.5–1.0; yellow,1.0–1.5; orange,1.5–2.0; red,2.0–5; pink,> 5 Cobalamin,propane-1,2-diol and K + are shown as ball-and-stick models (D) Stereo drawing of the distribution of the conserved and different residues Identical and different residues are shown in a blue ball-and-stick model and colored CPK models,respectively Red,different; orange,weakly conserved; yellow,strongly conserved [63].
Trang 8complex (2.50 A˚) whose structure was determined at 4C
[9] and significantly longer than those in the complexes of
diol dehydratase with cyanocobalamin (2.18 A˚) and with
adeninylpentylcobalamin (2.22 A˚) [10] We assigned the
former as the diol dehydrataseÆcob(II)alamin complex,
because no electron density corresponding to the cyano
group was observed [10] It has been reported that the
Co–CN bond is cleaved by X-ray irradiation during data
collection with diol dehydratase [58] Kratky and
cowork-ers have reported that free and glutamate mutase-bound
cyanocobalamin is reduced to cob(II)alamin by X-ray
irradiation [59] Therefore,we believe that the structure
reported in this paper is also that of the glycerol
dehydrataseÆcob(II)alamin complex The dihedral angle of
the northern and southern least-squares planes is 5.5,
indicating that the corrin ring of the glycerol
dehydratase-bound cobalamin is also almost planar,as compared with
that of free cyanocobalamin (14.1) This value is close to
those in the diol dehydratase-bound cobalamins (2.9–5.1)
[9,10]
Figure 4C indicates the comparison of the position of the
a-acetamide side chain of pyrrole ring A of the corrin ring in
the glycerol dehydratase-bound cobalamin (Fig 4Ca) with
those in the diol dehydratase-bound cobalamins It is clear that the direction of the a-acetamide side chain is very close
to that in the structure of the diol dehydrataseÆcobalamin complex determined at 4C [9] (Fig 4Cb) It seems that this side chain turns to the opposite direction to the cobalt atom, depending upon the steric bulk of the upper axial ligand (CN– or adeninylpentyl group) (Fig 4Cc,d) Thus, this offers evidence that the glycerol dehydratase-bound cobal-amin exists in a five-coordinated,square-pyramidal com-plex,suggesting again that the bound cobalamin is actually cob(II)alamin
Substrate- and K+-binding sites Substrate propane-1,2-diol and the essential cofactor K+
are bound inside the TIM barrel of the a subunit (Fig 4A) This suggests that K+bound in the active site of glycerol dehydratase in the presence of substrate is also not exchangeable with NH4+in the crystallization solution,as
in diol dehydratase [9] The two hydroxyl groups of substrate directly coordinate to K+ (Fig 5A) The O(2) and O(1) atoms of the substrate are fixed in the active site by hydrogen bonding with Glua171 and Glna297 and Hisa144
Fig 4 Structures of the active site and the
cobalamin-binding site (A) Stereo drawing of
the active-site cavity viewed from the direction
parallel to the plane of the corrin ring
Active-site residues interacting with the substrate
(green) and K + (cyan) are shown in
ball-and-stick models Cobalamin,pink (B) Residues
hydrogen-bonded to cobalamin The residues
interacting with cobalamin from distances
shorter by 0.1 A˚ and longer by 0.1 A˚ than
those in diol dehydratase are colored in yellow
and green,respectively The label B after the
residue number refers to residues of the b
subunit The red hydrogen bond is present
only glycerol dehydratase (C) Orientation of
the a-acetamide side chain of pyrrole ring A of
the corrin ring A possible hydrogen bond
between the a-acetamide side chain and an
amino acid residue is also shown (a) Glycerol
dehydrataseÆcobalamin complex (b) Diol
dehydrataseÆcobalamin complex whose
struc-ture was determined at 4 C (PDB: 1DIO) [9].
(c) Diol dehydrataseÆcyanocobalamin complex
(PDB: 1EGM) [10] (d) Diol
dehydra-taseÆadeninylpentylcobalamin complex (PDB:
1EEX) [10].
Trang 9and Aspa336,respectively (Fig 4A) K+is
hepta-coordi-nated by the two hydroxyls of the substrate and five oxygen
atoms of the active-site residues Such characteristics of the
substrate- and K+-binding sites are quite similar to those
seen in diol dehydratase [9] Although racemic
propane-1,2-diol was used for purification and crystallization,the
(R)-enantiomer is better fitted to the electron-density map
(Fig 5A) When R-values were compared with (R)- and
(S)-isomers in the active site,the (R)-isomer gave slightly
lower values Furthermore,when Fo) Fc maps were
compared,there was no significant electron density left for
the (R)-isomer,while slight electron density remained for the
(S)-isomer Thus,we assigned the (R)-isomer to the
electron-density map The kinetic results,however,indicate that
glycerol dehydratase shows almost equal affinity toward the
(S)- and (R)-isomers (Table 3) The reason for this
discrep-ancy is at present not clear In contrast,diol dehydratase
prefers the (S)-isomer (Km(R)/Km(S)¼ 3.1–3.2) [9] The
subtle differences between glycerol and diol dehydratases
in the positions of Vala301,Sera302,and Aspa336 (Fig 5B)
might explain the less marked preference of glycerol
dehydratase to the (S)-enantiomer in the substrate binding
Glycerol serves as a very good substrate as well as a potent
suicide inactivator for both glycerol dehydratase [39] and
diol dehydratase [3] It is well known that diol dehydratase
undergoes the inactivation by glycerol at a faster rate than
glycerol dehydratase [39,60] It was reported by Bachovchin
et al.that diol dehydratase distinguishes between R and S
binding conformations,the enzymeÆ(R)-glycerol complex
being predominantly responsible for the product-forming
reaction,while the enzymeÆ(S)-glycerol complex results
primarily in the inactivation reaction [60] Therefore,the
less marked preference of the glycerol dehydratase toward
the (S)-isomer explains why it is inactivated by glycerol
during catalysis at a slower rate than the diol dehydratase
A C K N O W L E D G M E N T S
We would like to thank Dr Keiko Miura for her kind help in data collection at the BL40B2 beamline,SPring-8,Japan We thank Ms Yukiko Kurimoto for her assistance in manuscript preparation.
R E F E R E N C E S
1 Banerjee,R.,ed (1999) Chemistry and Biochemistry of B 12 John Willy & Sons,New York.
2 Lee,H.A Jr & Abeles,R.H (1963) Purification and properties of dioldehydrase,an enzyme requiring a cobamide coenzyme J Biol Chem 238,2367–2373.
3 Toraya,T.,Shirakashi,T.,Kosuga,T & Fukui,S (1976) Coen-zyme B 12 -dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator Biochem Biophys Res Com-mun 69,475–480.
4 Abeles,R.H (1979) Current status of the mechanism of action of
B 12 -coenzyme In Vitamin B 12 (Zagalak,B & Friedrich,W.,eds),
pp 373–388 Walter de Gruyter,Berlin.
5 Toraya,T & Fukui,S (1982) Diol dehydrase In B 12 (Dolphin, D.,ed.),Vol 2,pp 233–262 John Wiley & Sons,New York.
6 Toraya,T (1994) Diol dehydrase and glycerol dehydrase,coen-zyme B 12 -dependent isozymes Metal Ions Biol Sys 30,217–254.
7 Toraya,T (1999) Diol dehydratase and glycerol dehydratase In Chemistry and Biochemistry of B 12 (Banerjee,R.,ed.),pp 783–809 John Wiley & Sons,New York.
8 Toraya,T (1998) Recent structure-function studies of B 12
coenzymes in diol dehydrase In Vitamin B 12 and B 12 -Proteins (Kra¨utler,B.,Arigoni,D & Golding,B.T.,eds),pp 303–320 Wiley-VCH,Weinheim.
9 Shibata,N.,Masuda,J.,Tobimatsu,T.,Toraya,T.,Suto,K., Morimoto,Y & Yasuoka,N (1999) A new mode of B 12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase Structure 7,997–1008.
10 Masuda,J.,Shibata,N.,Morimoto,Y.,Toraya,T & Yasuoka,
N (2000) How a protein generates a catalytic radical from
Fig 5 Interaction of glycerol dehydratase with the substrate (A) Stereo drawing of a part of the electron-density map (omit map,2F o ) F c ) contoured for the substrate and nearby amino acid residues K + and propane-1,2-diol are shown as a cyan CPK model and a green stick model,respectively (B) Stereo drawing
of the substrate and nearby residues Colored stick model,glycerol dehydratase; gray stick model,diol dehydratase.
Trang 10coenzyme B 12 : X-ray structure of diol
dehydratase-adeni-nylpentylcobalamin complex Structure 8,775–788.
11 Toraya,T.,Yoshizawa,K.,Eda,M & Yamabe,T (1999) Direct
participation of potassium ion in the catalysis of coenzyme B 12
-dependent diol dehydratase J Biochem 126,650–654.
12 Toraya,T.,Eda,M.,Kamachi,T & Yoshizawa,K (2001)
Energetic feasibility of hydrogen abstraction and recombination in
coenzyme B 12 -dependent diol dehydratase reaction J Biochem.
130,865–872.
13 Eda,M.,Kamachi,T.,Yoshizawa,K & Toraya,T (2002)
The-oretical study on the mechanism of catalysis of coenzyme B 12
-dependent diol dehydratase Bull Chem Soc Jpn 75,1469–1481.
14
Mancia,F.,Keep,N.H.,Nakagawa,A.,Leadlay,P.F.,Mcswee-ney,S.,Rasmussen,B.,Bo¨secke,P.,Diat,O & Evans,P.R (1996)
How coenzyme B 12 radicals are generated: the crystal structure of
methylmalonyl-coenzyme A mutase at 2 A˚ resolution Structure 4,
339–350.
15 Reitzer, R., Gruber, K., Jogl, G., Wagner, U.G., Bothe, H.,
Buckel,W & Kratky,C (1999) Glutamate mutase from
Clos-tridium cochlearium: the structure of a coenzyme B 12 -dependent
enzyme provides new mechanistic insights Structure 7,891–902.
16 Sintchak,M.D.,Arjara,G.,Kellogg,B.A.,Stubbe,J & Drennan,
C (2002) The crystal structure of class II ribonucleotide reductase
reveals how an allosterically regulated monomer mimics a dimer.
Nat Struct Biol 9,293–300.
17 Toraya,T (2000) Radical catalysis of B 12 enzymes: structure,
mechanism,inactivation,and reactivation of diol and glycerol
dehydratases Cell Mol Life Sci 57,106–127.
18 Toraya,T (2002) Enzymatic radical catalysis: coenzyme B 12
-dependent diol dehydratase Chem Rec in press.
19 Pawelkiewicz,J & Zagalak,B (1965) Enzymatic conversion of
glycerol into b-hydroxypropionaldehyde in a cell-free extract from
Aerobacter aerogenes Acta Biochim Polon 12,207–218.
20 Schneider,Z.,Larsen,E.G.,Jacobson,G.,Johnson,B.C &
Pawelkiewicz,J (1970) Purification and properties of glycerol
dehydrase J Biol Chem 245,3388–3396.
21 Stroinski,A.,Pawelkiewicz,J & Johnson,B.C (1974) Allosteric
interactions in glycerol dehydratase Purification of enzyme and
effects of positive and negative cooperativity for glycerol Arch.
Biochem Biophys 162,321–330.
22 Toraya,T & Fukui,S (1977) Immunochemical evidence for the
difference between coenzyme-B12-dependent diol dehydratase and
glycerol dehydratase Eur J Biochem 76,285–289.
23 Toraya,T.,Kuno,S & Fukui,S (1980) Distribution of coenzyme
B12-dependent diol dehydratase and glycerol dehydratase in
selected genera of Enterobacteriaceae and Propionibacteriaceae.
J Bacteriol 141,1439–1442.
24 Forage,R.G & Foster,M.A (1979) Resolution of the coenzyme
B-12-dependent dehydratases of Klebsiella sp & Citrobacter
freundii Biochim Biophys Acta 569,249–258.
25 Toraya,T.,Honda,S.,Kuno,S & Fukui,S (1978) Coenzyme
B 12 -dependent diol dehydratase: regulation of apoenzyme
synth-esis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724.
J Bacteriol 135,726–729.
26 Forage,R.G & Foster,M.A (1982) Glycerol fermentation in
Klebsiella pneumoniae: functions of the coenzyme B12-dependent
glycerol and diol dehydratases J Bacteriol 149,413–419.
27 Forage,R.G & Lin,E.C (1982) dha system mediating aerobic
and anaerobic dissimilation of glycerol in Klebsiella pneumoniae
NCIB 418 J Bacteriol 151,591–599.
28 Tobimatsu,T.,Azuma,M.,Matsubara,H.,Takatori,H.,Niida,
T.,Nishimoto,K.,Satoh,H.,Hayashi,R & Toraya,T (1996)
Cloning,sequencing and high-level expression of the genes
encoding adenosylcobalamin-dependent glycerol dehydrase of
Klebsiella pneumoniae J Biol Chem 271,22352–22357.
29 Seyfried,M.,Daniel,R & Gottschalk,G (1996) Cloning,
sequencing,and overexpression of the genes encoding coenzyme
B 12 -dependent glycerol dehydratase of Citrobacter freundii.
J Bacteriol 178,5793–5796.
30 Hosoi,N.,Morimoto,K.,Ozaki,C.,Kitamoto,Y & Ichikawa,
Y (1978) Enzyme activities involved in the metabolism of 1,2-propanediol by Propionibacterium freudenreichii J Ferment Techn 56,566–572.
31 Toraya,T.,Honda,S & Fukui,S (1979) Fermentation of 1,2-propanediol and 1,2-ethanediol by some genera of Enterobacteriaceae,involving coenzyme B 12 -dependent diol dehydratase J Bacteriol 139,39–47.
32 Tobimatsu,T.,Hara,T.,Sakaguchi,M.,Kishimoto,Y.,Wada, Y.,Isoda,M.,Sakai,T & Toraya,T (1995) Molecular cloning, sequencing and expression of the genes encoding adenosylcoba-lamin-dependent diol dehydrase of Klebsiella oxytoca J Biol Chem 270,7142–7148.
33 Bobik,T.A.,Xu,Y.,Jeter,R.M.,Otto,K.E & Roth,J.R (1997) Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase J Bacteriol 179, 6633–6639.
34 Tobimatsu,T.,Azuma,M.,Hayashi,S.,Nishimoto,K & Toraya,
T (1998) Molecular cloning,sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae Biosci Biotechn Biochem 62,1774–1777.
35 Tobimatsu,T.,Sakai,T.,Hashida,Y.,Mizoguchi,N.,Miyoshi,S.
& Toraya,T (1997) Heterologous expression,purification,and properties of diol dehydratase,an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca Arch Biochem Biphys 347,132– 140.
36 Schneider,Z.,Pech,K & Pawelkiewicz,J (1966) Enzymic transformation of glycerol to beta-hydroxypropionic aldehyde II Dissociation of the enzyme into two protein fragments Bull Acad Pol Sci 14,7–12.
37 Toraya,T.,Ushio,K.,Fukui,S & Hogenkamp,H.P.C (1977) Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme.
J Biol Chem 252,963–970.
38 Toraya,T.,Krodel,E.,Mildvan,A.S & Abels,R.H (1979) Role
of peripheral side chains of vitamin B 12 coenzymes in the reaction catalyzed by dioldehydrase Biochemistry 18,417–426.
39 Poznanskaya,A.A.,Yakusheva,M.I & Yakovlev,V.A (1977) Study of the mechanism of action of adenosylcobalamin-depen-dent glycerol dehydratase from Aerobacter aerogenes II The inactivation kinetics of glycerol dehydratase complexes with adenosylcobalamin and its analogs Biochim Biophys Acta 484, 236–243.
40 Lowry,O.H.,Rosebrough,N.J.,Farr,A.L & Randall,R.J (1951) Protein measurement with the folin phenol reagent J Biol Chem 193,265–275.
41 Gill,S.C & Von Hippel,P.H (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Bio-chem 182,319–326.
42 Davis,B.J (1964) Disc electrophoresis-II: method and application
to human serum proteins Ann N Y Acad Sci 121,404–427.
43 Laemmli,U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.
44 Lambin,P & Fine,J.M (1979) Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents Anal Biochem 98, 160–168.
45 Toraya,T & Ishida,A (1991) Roles of the D -ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adeno-sylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction J Biol Chem 266,5430–5437.
46 Yamanishi,M.,Yamada,S.,Muguruma,H.,Murakani,Y., Tobimatsu,T.,Ishida,A.,Yamauchi,J & Toraya,T (1998) Evidence for axial coordination of 5,6-dimethylbenzimidazole to