From the
Chitin synthase 2 of Saccharomyces cerevisiae was
characterized by means of site-directed mutagenesis and subsequent
expression of the mutant enzymes in yeast cells. Chitin synthase 2
shares a region whose sequence is highly conserved in all chitin
synthases. Substitutions of conserved amino acids in this region with
alanine (alanine scanning) identified two domains in which any
conserved amino acid could not be replaced by alanine to retain enzyme
activity. These two domains contained unique sequences,
Glu
Chitin, a
Chs1p, the most abundant enzyme in
S. cerevisiae cells among three chitin synthases, is
solubilized from a particulate fraction with digitonin and is partially
purified through gel filtration followed by product
entrapment
(11) . Molecular mass of the partially purified Chs1p
is about 570 kDa in which a 63-kDa polypeptide is a major component of
the enzyme
(11) . The enzyme requires UDP-GlcNAc as the
substrate
(10, 11) , and although GlcNAc is an activator
of the enzyme, the real role of GlcNAc remains to be
established
(10, 11) . Chs2p has been characterized in
the membrane fraction of S. cerevisiae cells lacking CHS1 and is shown to be much less abundant than Chs1p (the Chs2p level
is estimated to be about 5% of that of Chs1p)
(12) . Although
Chs1p and Chs2p share sequence similarity and many characteristics such
as membrane localization, activation by GlcNAc, use of UDP-GlcNAc as
the substrate, and K
Despite the characterization of chitin synthase activities
and cloning of the chitin synthase genes, no information is available
about the protein domains essential for enzyme activity such as active
sites, probably due to the difficulties in overexpressing and purifying
the enzyme in large quantities. Here we report the highly conserved
region of Chs2p and the possible involvement of this region in the
catalytic reaction by chitin synthase is proposed.
A
0.8-kb
When the amino acid sequence of S. cerevisiae Chs2p
was compared to those of other chitin synthases, we found that a region
comprising amino acid positions 490-607 was highly conserved in
all types of chitin synthases of yeasts as well as mycelial fungi
(Fig. 1A; see also Refs. 2-8 and 28). This region
was, therefore, designated as Con1 (conserved region 1). Con1 could be
divided into three subdomains based on the frequencies of the
appearance of conserved amino acids (Fig. 1B). The
sequence of Chs3p became rather diverse in the N-terminal region
outside Con1, whereas Chs1p and Chs2p still shared high sequence
similarity. Further N-terminal and C-terminal regions outside Con1
showed much lower sequence similarity even between Chs1p and Chs2p.
Since Chs1p, 2p, and 3p perform the same catalytic function, the active
site of these enzymes would be expected to be conserved. From this
point of view, it is likely that Con1 contains the active site of
chitin synthase. In order to address this possibility, we generated a
series of mutant enzymes in which one of the conserved amino acids in
Con1 was replaced by alanine.
Activities of each mutant enzyme were determined using total
membrane fractions. As shown in Fig. 2, substitution of
Tyr
Next, we examined whether or not the
hydroxyl group of Asp
The above results
strongly suggest that Glu
We demonstrated here that Asp
If Asp
Interestingly, similar sequences to
Glu
We thank Dr. O. Shimmi for valuable suggestions and
repeated discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
,
that were conserved in all types of chitin synthases. Glu
or arginine at 563, 602, and 603 could be substituted by glutamic
acid and lysine, respectively, without significant loss of enzyme
activity. However, even conservative substitutions of Asp
with glutamic acid, Gln
with asparagine,
Arg
with lysine, or Trp
with tyrosine
drastically decreased the activity, but did not affect apparent
K
values for the substrate significantly.
In addition to these amino acids, Asp
was also found in
all chitin synthase. The mutant harboring a glutamic acid substitution
for Asp
severely lost activity, but it showed a similar
apparent K
value for the substrate.
Amounts of the mutant enzymes in total membranes were more or less the
same as found in the wild type. Furthermore, Asp
,
Asp
, Gln
, Arg
, and
Trp
are completely conserved in other proteins possessing
N-acetylglucosaminyltransferase activity such as NodC proteins
of Rhizobium bacterias. These results suggest that
Asp
, Asp
, Gln
,
Arg
, and Trp
are located in the active
pocket and that they function as the catalytic residues of the enzyme.
-1,4-linked polymer of
N-acetylglucosamine, is one of the components of the yeast
cell wall and is synthesized by the enzyme called chitin
synthase
(1) . Three chitin synthase genes have been identified
in the yeasts Saccharomyces
cerevisiae(2, 3, 4, 5) and
Candida albicans(6, 7, 8) , and they
are designated as CHS1, CHS2, and CHS3 (CHS3 is called as CAL1/CSD2 in S.
cerevisiae)
(2, 3, 4, 5, 6, 7, 8, 9) .
Chitin synthase 1 (Chs1p) and Chs2p are
zymogens
(10, 11, 12) , but Chs3p is not simply
activated by proteinases
(4, 13, 14) ; Chs3p
shows zymogenic property only when it is treated with proteinases in
the presence of the substrate, UDP-GlcNAc
(15) . Several lines of
evidence, including the phenotypes of cells in which one of the chitin
synthase genes is disrupted, suggest a functional distinction for each
chitin synthase in S. cerevisiae; Chs1p repairs damaged chitin
during cell separation, Chs2p is required for primary septum formation,
and Chs3p is involved in all other chitin
syntheses
(16, 4, 17) . Although none of chitin
synthase genes is so far reported to be essential for the vegetative
growth of yeast cells
(2, 17, 18) , combinational
disruption of CHS2 and CAL1 is lethal in S.
cerevisiae(17) , suggesting that Chs1p can not functionally
complement Chs2p and Chs3p.
for the substrate,
they show different specificity for divalent cations and different pH
optima
(10, 12) . CHS1 seems not to be a
critical gene for chitin synthesis in yeast cells because S.
cerevisiae cells lacking CHS1 grow normally without
showing apparent loss of cell wall chitin content in the absence of
Chs1p activity
(2) . On the other hand, disruption of CHS2 causes severe growth defects and morphological
abnormalities
(17) , suggesting that Chs2p plays more important
roles in chitin synthesis in vivo and in cell growth than
Chs1p.
Yeast Strains and Plasmids
Haploid S.
cerevisiae strain, R27-7C-1C (a trp1 leu2 ura3
his3), was used as the wild type strain. RRA400 (a trp1 leu2
ura3 his3 cal1::HIS3) was a derivative of R27-7C-1C in
which CAL1 was replaced by HIS3 using YIp plasmid
(19) carrying CAL1 and HIS3. In RRA400,
CHS1 was further replaced by URA3 using YIp plasmid
(19) carrying CHS1 and URA3 to generate
RRA400-1U (a trp1 leu2 ura3 his3 chs1
::URA3
cal1
::HIS3) which harbored neither CHS1 nor
CAL1. Cells of the above strains were cultured at 30 °C
with yeast nitrogen base supplemented with glucose and required amino
acids. Expression of chitin synthase was induced by transferring the
cells in early logarithmic phase to the medium containing galactose and
further culturing the cells for 12 h at 30 °C.
(
)BamHI-EcoRI fragment
containing GAL1 promoter and a 0.2- kb
XbaI-HindIII fragment containing GAP terminator were excised from pYPR3831
(20) and ligated at
the HindIII cleavage site of YEp351
(21) in which
unique SalI site had been already destroyed. The resulting
plasmid harboring GAL1 promoter, GAP terminator,
2-µm replication origin, and LEU2 as a selectable marker
was designated as YpLX. To overexpress Chs2p in S. cerevisiae cells, CHS2 was introduced downstream of the GAL1 promoter. 2.9-kb DNA fragment containing the entire coding region
of S. cerevisiae CHS2 was amplified by polymerase chain
reaction. The primers used for the polymerase chain reaction were
5`-GACTCTAGAATGACGAGAAACCCG-3` and
5`-GATGCGGCATCTAGATTAGCCCTTTTTGTGGAA-3` which possessed XbaI
cleavage site in addition to the corresponding sequences to
CHS2. The resulting DNA was ligated at the XbaI
cleavage site of YpLX which was located at the junction of GAL1 promoter and terminator. The resulting plasmid, YpLCS2, was then
transfected into the yeast cells by electroporation as
described
(22) , and leucine prototrophs were collected and used
for the experiments.
Site-directed Mutagenesis
A series of CHS2 mutants were generated by site-directed mutagenesis using
uracil-containing single-stranded DNA as described by Kunkel et
al.(23, 24) . A 1.6-kb PstI/SalI
fragment of CHS2 was cloned in pUC118 and used for generating
uracil-containing single stranded DNA. DNA fragments of CHS2 harboring the particular mutations were ligated at
PstI-SalI cleavage site of YpLCS2 and transfected
into S. cerevisiae cells. All mutations were confirmed by
sequencing the DNA as described elsewhere
(25) .
Assay of Chitin Synthase
S. cerevisiae cells with or without the induction of chitin synthases were
harvested and washed twice with 20 mM Tris-Cl, pH 7.5, and
suspended in 1 ml/g yeast of a buffer containing 20 mM
Tris-Cl, pH 7.5, 0.25 mM phenylmethylsulfonyl fluoride, 2
µg/ml chymostatin, 1.5 µg/ml leupeptin, 1 µg/ml pepstatin,
and 5 µg/ml antipain. Cells were lysed with glass beads, and cell
debris were removed by the low speed centrifugation. The membrane was
then sedimented at 100,000 g for 50 min at 4 °C,
washed once with 20 mM Tris-Cl, pH 7.5, suspended in a buffer
containing 20 mM Tris-Cl, pH 7.5, and 33% glycerol, and stored
at -80 °C until use. Chs2p assay was carried out according to
the method of Sburlati and Cabib
(12) in a standard 100-µl
reaction mixture containing 30 mM Tris-Cl, pH 7.5, 2.5
mM Co(CH
COO)
, 32 mM GlcNAc,
0.1 mM [
H]UDP-GlcNAc (specific activity,
95,880 disintegrations/min/nmol), 10-50 µg of protein of
yeast membrane fraction at 30 °C for 60 min.
Generation of Antibody
A polyclonal antibody was
raised against a part of Chs2p (from amino acid positions 192 to 624)
which was expressed in insect cells (Sf21 cells) as a fusion protein
with a 6-histidine tail
(26) . In this fusion protein, the 6
histidines were linked to the C-terminal end. A 1.3-kb fragment of
CHS2 encoding 433 amino acids (from amino acid positions
192-624 of Chs2p) was amplified by polymerase chain reaction
using CHS2 gene as a template, and ligated at the
BamHI-XhoI cleavage site of pBacPAK9
(27) .
Primers used for amplifying a part of CHS2 gene were
5`-CGGCGGATCCAAATGTCTGCAGACACTTTCAATGAAACA-3` and
5`-CCGGCCTCGAGCCAAATTTGGTAGAAATGCAATTGAGCA-3`. The resulting
plasmid harboring a part of CHS2 gene was then digested with
XhoI and ligated with an oligonucleotide coding for 6
histidines to generate pB9CS2(192-624)His.
pB9CS2(192-624)His was transfected into Sf21 cells together with
BacPAK6 viral DNA using lipofectin
(27) . Sf21 cells were
harvested 72 h post-infection, and the insoluble Chs2p-6-histidine
fusion protein was extracted by lysing cells with 6 M
guanidine-HCl, purified by Nickel chelating column chromatography
through a linear gradient of immidazole (0-500 mM) in 6
M guanidine-HCl, precipitated by trichloroacetic acid, and
injected into rabbits subcutaneously. IgG fractions containing
anti-Chs2p antibody were obtained from crude sera by ammonium sulfate
precipitation followed by Protein A-Superose column chromatography.
Western Blotting
20 µg of protein of total
yeast membranes prepared as mentioned above were fractionated on 8%
SDS-polyacrylamide gel, transferred to a PVDF membrane
electrophoretically, Western blotted with anti-Chs2p polyclonal
antibody, and then with horseradish peroxidase-conjugated anti-rabbit
IgG. Chs2p was visualized by incubating a PVDF membrane with cyclic
diacylhydrazides (ECL detection kit, Amersham) and subsequent exposure
to an x-ray film (Kodak). Quantification of Chs2p in the total membrane
was carried out by comparing the density of Chs2p in the total membrane
with that of the purified Chs2p expressed in Sf21 cells using a
densitometer.
Figure 1:
Highly conserved region of Chs2p.
A, location of highly conserved region of Chs2p. Con1 represents the highly conserved region of Chs2p with brackets indicating subdomains in this region. Numbers in the
upper side of the bar indicate the position of amino acids from the
N-terminal end of Chs2p. B, comparison of the amino acid
sequence of Con1 with several chitin synthases. Amino acids drawn with
bold characters are those conserved in all chitin synthases
listed here. I-III represent subdomains where conserved
amino acids appear at high frequencies. TM, potential
transmembrane domains; ScChs1-3, S.
cerevisiae chitin synthase 1-3; CaChs1-3,
C, albicans chitin synthase 1-3;
RoChs1-3, R. oligosporus chitin
synthase 1-3 (40); AnChsA, B, A. nidulans chitin synthase A, B.
Chs2p could not be expressed in
bacterial or insect cells in an active form. Therefore, a series of
plasmid DNAs carrying mutant CHS2 genes whose expression was
under the control of GAL1 promoter was introduced into the
yeast strain, RRA400-1U, in which endogenous CHS1 and
CAL1 had been disrupted, and expression of the mutant enzymes
were induced by culturing the cells in medium containing galactose.
When YpLCS2 was introduced into RRA400-1U, Chs2p activity was
completely dependent on galactose, and endogenous Chs2p activity was
barely detected, indicating that the endogenous Chs2p activity was
extremely low and could be neglected in this system (data not shown).
, Glu
, Asp
,
Arg
, Gln
, Arg
,
Arg
, Arg
, or Trp
with alanine
resulted in almost complete loss of the activity, whereas alanine
substitution of other amino acids retained activities. It was also
demonstrated in the control experiments that the amino acids which were
not highly conserved (Asn
, Ser
,
Asp
, Asp
, Asn
,
Arg
, His
) could be substituted by alanine
without affecting the enzyme activity significantly (Fig. 2).
Figure 2:
Effects of amino acid substitutions in
Con1 on the activity of Chs2p. Conserved amino acids in Con1 were
substituted by alanine, and the effect on enzyme activity is shown in
the left panel. The effect of substituting non-conserved amino
acids with alanine also are indicated in the right panel.
Activities of these mutant enzymes were determined in a standard
reaction mixture using 10 µg of the total membrane protein at 30
°C for 60 min.
Domains II and III contained unique sequences,
Glu-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
,
respectively. These two sequences are completely conserved in all
chitin synthases listed in Fig. 1including newly identified
chitin synthases of Rhizopus oligosporus and Aspergillus
nidulans. The same sequences were also found in recently reported
Neurospora crassa Chs1p and Chs2p
(28) , suggesting that
they are critical sites for the catalytic activities of chitin
synthases. In order to address this possibility, we have substituted
the amino acids found in these sequences with the analogous ones. As
mentioned above, no amino acid in these sequences could be replaced by
alanine to retain activity, but E561D, R563K, R602K, and R603K did
retain activity, suggesting that Glu
, Arg
,
Arg
, and Arg
, while quite important for
enzyme activity, may not be in the active site itself. However, even
conservative replacements in D562E, Q601N, R604K, and W605Y had very
little activity (). With the exception of D562E, which was
too low to determine the apparent K
value, conservative substitutions for any other amino acid in
Glu
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
did not affect their apparent K
values for the substrate significantly (). Even
Q601N, R604K, and W605Y exhibited similar apparent
K
values to that of wild type enzyme
despite that enzyme activities of these mutants were about 1% of the
wild type enzyme activity.
was necessary for the catalytic
activity of the enzyme. Substitution of asparagine for Asp
resulted in the complete loss of the activity ().
Interestingly, E561Q also showed very little activity (),
indicating that the hydroxyl groups of both Glu
and
Asp
are essential for the enzyme activity. Although
individual residues in the stretch of basic arginines in domain III
(Arg
or Arg
) could be replaced by lysine
without apparent loss of the enzyme activity, we wondered whether both
of these basic amino acids could be replaced by lysine simultaneously.
The mutant enzyme harboring lysine substitution of Arg
and Arg
together lost the enzyme activity. This
result implies that Arg
and Arg
are not
located simply to provide basic charges and suggests that they may play
some significant roles in catalytic activity.
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
are essential sites for the catalytic activity and that some of
the amino acids in these sequences, specifically Asp
,
Gln
, Arg
and Trp
, may
function as the catalytic site of the enzyme. However, we cannot rule
out the possibility that a decrease in the activities of the mutant
enzymes was the trivial consequence of the lower levels of expression.
To address this possibility, a polyclonal antibody was raised against
Chs2p (from amino acids 192-624) to examine levels of the
expression of all of these mutant enzymes. Western blotting revealed
that this antibody recognized a protein with approximately molecular
mass of 110 kDa in the membranes prepared from cells overexpressing
CHS2 but not in the membranes from the vector-transfected
control (Fig. 3). The size of this protein corresponded to that
estimated by the deduced amino acid sequence of Chs2p. Furthermore,
this antibody precipitated the chitin synthase activity when incubated
with partially purified Chs2p by product entrapment (data not shown).
All these results indicate that the 110-kDa protein detected by the
Western blotting was actually Chs2p. Using a part of Chs2p (from amino
acids 192-624) purified from insect cells as a standard, the
amount of Chs2p in the total yeast membranes of RRA400-1U
harboring YpLCS2 was estimated to be about 60 pg/µg of membrane
protein when Chs2p expression was induced by galactose. Western blots
of total membranes prepared from cells overexpressing mutant Chs2p
revealed no significant difference in amounts of protein between wild
type and a series of the mutants except for Y521A (Fig. 4, A and B). The expression level of Y521A was about 20% of
that of the wild type enzyme, and this might account for a portion of
the reduced activity of Y521A ( Fig. 2and
Fig. 4A). These results demonstrate that except for
Y521A a decrease in the activity of mutant enzymes was the consequence
of decrease in the rate of catalytic reaction, and that no other
mutation in Chs2p reduced the level of protein expressed. Considering
all the data mentioned above, we conclude that the conserved
Glu
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
regions of Chs2p are essential for catalytic activity and that
Asp
, Gln
, Arg
, and
Trp
are the potential catalytic residues of the enzyme.
Figure 3:
Expression of Chs2p in insect cells and
generation of antibody against Chs2p. A, the Chs2p protein
fragment (from amino acid positions 192-624) was isolated from
Sf21 cells as described under ``Materials and Methods.'' The
8% SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue.
Lane 1, total extract of uninfected Sf21 cells; lane
2, total extract of infected Sf21 cells; lane 3, purified
Chs2p fragment. B, 20 µg of membrane protein prepared from
cells of RRA400-1U which were transfected with YpLX (vector) or
with YpLCS2 (plasmid carrying CHS2) were fractionated on 8%
SDS-polyacrylamide gels, transferred to a PVDF membrane, and visualized
by Western blotting with anti-Chs2p antibody. Lane 1, purified
Chs2p fragment; lane 2, total membrane of RRA400-1U
harboring YpLX; lane 3, total membrane of RRA400-1U
harboring YpLCS2.
Figure 4:
Comparison of levels of protein in wild
type and mutant Chs2p constructs. 20 µg of membrane protein from
cells overexpressing wild type as well as mutant Chs2p were subjected
to 8% SDS-polyacrylamide gel electrophoresis. After blotting on to a
PVDF membrane, Chs2p was visualized by Western blotting. Only the Chs2p
bands were indicated. Additional details are under ``Materials and
Methods.'' A, levels of mutant Chs2p in which one of the
conserved amino acids in Con1 was substituted by alanine. B,
levels of mutant Chs2p in which one or two of the amino acids in
Glu-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
were conservatively substituted. C, levels of mutant
Chs2p in which Asp
was substituted with the indicated
amino acid.
GlcNAc is known to be an activator of chitin synthases; millimolar
addition of GlcNAc to the reaction mixture increases the enzyme
activity by severalfold
(9, 11, 12) . We have
tested the effect of GlcNAc on some of the mutant enzymes. Enhancement
of the activity by GlcNAc was quite high in R563K and R602K
(Fig. 5B), whereas those of E561D, Q601N, R603K, R604K,
and W605Y were not significantly different from wild type enzyme
(Fig. 5A). Although the rate of the activation by GlcNAc
was much higher in R602K than in R563K, 240 mM GlcNAc
increased the enzyme activity of R563K and R602K by more than 20-fold.
At this concentration of GlcNAc (240 mM), only about 5-fold
increase in the activity was observed with wild type enzyme
(Fig. 5B). The finding that mutations in the two
separate regions made the enzymes sensitive to enhanced activation by
the same small molecule, GlcNAc, implies that these two region
Glu-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
may be located very close to each other three dimensionally.
Figure 5:
Effects of GlcNAc on the activity of
Chs2p. Total membranes were prepared from cells of RRA400-1U
transfected with YpLCS2 that harbored indicated mutations of CHS2 gene, and Chs2p activity was determined using 10 µg of
membrane protein in a standard reaction mixture containing indicated
concentrations of GlcNAc.
Recently, Szaniszlo and Momany
(29) reported that chitin
synthases share sequence homology with a short stretch of amino acids
constituting the nucleotide-binding fold of a bacterial periplasmic
permease (AraG) and of an adenylate kinase (Adk). In this small region,
one aspartic acid is completely conserved in all chitin synthases
(Asp in Chs2), AraG and Adk (Fig. 6A; see
also Ref. 29). These facts also prompted us to examine the possibility
that Asp
of Chs2p is a part of the active site.
Substitution of Asp
of Chs2p with alanine or glutamic
acid resulted in nearly a complete loss of the enzyme activity.
Although the apparent K
value of D441A
could not be determined because of its extremely low activity, that of
D441E was not significantly different from that of wild type enzyme,
and the amount of protein expressed of the above two mutant enzymes was
more or less the same as the control Chs2p (Fig. 4C and
). All these results indicate that Asp
is
also an essential amino acid for the catalytic reaction of the enzyme.
Figure 6:
Sequence similarity of chitin synthases
and NodC proteins. A, sequences of chitin synthases in the
region which is homologous to the nucleotide binding fold of a
bacterial periplasmic permease (AraG) and an adenylate kinase (Adk)
were compared with those of NodC proteins, cellulose synthase, and DG42
protein. The amino acid drawn in bold corresponds to
Asp of S. cerevisiae Chs2p. B,
sequences of chitin synthases in domains II and III within Con1 were
compared to those of NodC proteins, cellulose synthase, and DG42
protein. Amino acids in bold letters are those that correspond
to Glu
, Gln
, Arg
, and
Trp
of S. cerevisiae Chs2p. SacChs2,
S. cerevisiae Chs2p (3); CanChs1, C. albicans Chs1p (5, 6); SacChs1, S. cerevisiae Chs1p (2);
SacChs3, S. cerevisiae Cal1p (4); RlvNodC,
Rhizobium leguminosarum bv. viciae NodC (41);
RfNodC, Rhizobium fredii NodC (42); RmNodC,
Rhizobium melioti NodC (33); AcNodC, Azorhizobium
caulinodans NodC (43); SpHasA, Streptococcus pyogenes hyaluronan synthase (35, 36); AxCs, Acetobacter
xylinum cellulose synthase (37); XlDG42, X.
laevis DG42 protein (38).
,
Glu
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
of Chs2p play essential roles in the catalytic activity of the
enzyme, and that among them, Asp
, Asp
,
Gln
, Arg
, and Trp
are
potential catalytic residues. Catalytic mechanisms deduced for several
enzymes, including glycosidases and glycosyltransferases, suggest that
acidic or basic catalysts are involved in the protonation of the
substrate
(30) . In sucrose:1,3-
-D-glucan
3-
-D-glycosyltransferases (GTase I) of Streptococcus
sobrinus, the aspartic acid near the N-terminal end
(Asp
) is identified as a catalytic site and is thought to
function as an acidic catalyst
(31) . Since Asp
and
Asp
of Chs2p are conserved in all chitin synthases and
when substituted by asparagine do not retain enzyme activity, it is
very likely that one of these residues (Asp
or
Asp
) is functioning as an acidic catalyst. However, we
cannot rule out the possibility that Asp
is involved in
the substrate recognition and binding because activities of D562A,
D562E, and D562N were too low to determine their apparent
K
values for the substrate.
Interestingly, E561Q also showed very little activity (),
suggesting that the hydroxyl group of Glu
may be required
for the efficient ionization of the hydroxyl group of
Asp
, if Asp
indeed is functioning as an
acidic catalyst. Arg
and Arg
are also
required for activity; simultaneous substitution of these two arginines
with lysines resulted in the complete loss of the activity. Because
Arg
cannot be substituted even with lysine, it is
speculated that Arg
may be functioning as a basic
catalyst and that two arginines in front of Arg
may be
required for the efficient ionization of the guanidino group of
Arg
.
, Asp
,
Gln
, Arg
, and Trp
of Chs2p
are catalytic residues, they might be also conserved in proteins
possessing the same catalytic activity as chitin synthases. NodC
proteins of Rhizobium as well as Azorhizobium bacterias share significant sequence homology to chitin synthases
(Fig. 6). Further, catalytic activity and the substrate of NodC
protein are the same as those of chitin synthases
(32) . When
sequences of NodC proteins were compared to those of chitin synthases,
all NodC proteins were found to have sequences, Glu-Asp-Arg and
Gln-Gln-Leu-Arg-Trp, which were identical or quite similar to unique
sequences identified in chitin synthases (Glu-Asp-Arg and
Gln-Arg-Arg-Arg-Trp) (Fig. 6B; see also Refs. 33 and
34). In these sequences, amino acids essential for the catalytic
activity of Chs2p could not be substituted even conservatively, Asp in
Glu-Asp-Arg, Gln, the third Arg and Trp in Gln-Arg-Arg-Arg-Trp, were
completely conserved in NodC proteins as well. Further, Asp
of Chs2p is also found in NodC proteins (Fig. 6A,
and see also Ref. 29), although the neighboring sequences of this
aspartic acid are not highly conserved between chitin synthases and
NodC proteins. All these facts strongly support the idea that
Asp
,
Glu
-Asp
-Arg
, and
Gln
-Arg
-Arg
-Arg
-Trp
of Chs2p are crucial sites for catalytic activity and that among
them Asp
, Asp
, Gln
,
Arg
, and Trp
are the potential catalytic
residues of the enzyme.
-Asp
-Arg
and
Gln
-Arg
-Arg
-Arg
-Trp
of Chs2p were also found in hyaluronan synthase of
Streptococcus pyogenes(35, 36) , cellulose
synthase of Acetobacter xylinum(37) and DG42 of
Xenopus laevis(38) . These are
Asp
-Asp
-Arg
and
Gln
-Gln
-Asn
-Arg
-Trp
in hyaluronan synthase,
Tyr
-Asp
-Ala
and
Gln
-Arg
-Val
-Arg
-Trp
in cellulose synthase, and
Asp
-Asp
-Arg
and
Gln
-Gln
-Thr
-Arg
-Trp
in DG42, respectively (Fig. 6). As seen in NodC proteins,
Asp in Glu-Asp-Arg, Gln, the third Arg and Trp in Gln-Arg-Arg-Arg-Trp
of Chs2p were also conserved in corresponding regions of hyaluronan
synthase, cellulose synthase, and DG42 (Fig. 6, and see also Ref.
39). Although catalytic activity of DG42 remains to be established,
hyaluronan synthase and cellulose synthase catalyze the polymerization
of sugars with
-1,4-linkage using UDP-sugar as substrate. Thus,
amino acids of the potential catalytic sites of Chs2p may be generally
conserved in glycosyltransferases which catalyze the synthesis of
oligosaccharides with
-1,4-linkages.
Table:
Characteristics of mutant Chs2
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.