Biologia 63/6: 1028—1034, 2008
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-008-0163-3
Structure-function relationships in human salivary α-amylase:
role of aromatic residues in a secondary binding site
Chandran Ragunath, Suba G.A. Manuel, Chinnasamy Kasinathan
& Narayanan Ramasubbu*
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103 USA; e-mail:
[email protected]
Abstract: Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrolysis of starch;
(ii) binding to hydroxyapatite; and (iii) binding to bacteria (e.g. viridans streptococci). Oral bacteria utilize the starch
hydrolyzing activity of HSAmy to derive their nutrients from dietary starch. Localized acid production by bacteria, through
the metabolism of maltose generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries
formation. HSAmy is a component of the acquired enamel pellicle and is used by Streptococcus gordonii to colonize the
oral cavity. Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the
bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic
residues are prominently located, we hypothesized that one of the secondary saccharide-binding sites harboring the aromatic
residues W316 and W388, may play an important role in bacterial binding. To test this hypothesis, the aromatic residues
W316 and W388 were mutated to alanine. The wild type and the mutant enzymes were characterized for their abilities to
exhibit enzyme activity, starch binding and bacterial binding. Our results clearly showed that (i) the mutants W316A and
W388A were not impaired in starch binding or bacterial binding; (ii) mutation of aromatic residues at theses sites does not
alter the overall conformation of the molecule; and (iii) the hydrolytic activity of the enzyme is unaffected against starch
as substrates but reduced significantly against oligosaccharides.
Key words: salivary α-amylase; site directed mutagenesis; subsite engineering; oligosaccharide hydrolysis; crystal structure;
bacterial binding.
Introduction
α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC
3.2.1.1) are ubiquitous enzymes that catalyze the hydrolysis of starch and related polysaccharides containing α-1,4-glucosidic bonds. They belong to the glycoside hydrolase family 13 (MacGregor et al. 2001) and
are widespread in all three domains of life. In humans,
α-amylase is present in both salivary and pancreatic secretions and exhibits a high level of structural similarity (Brayer et al. 1995; Ramasubbu et al. 1996). Human
salivary α-amylase (HSAmy) is an important enzyme in
the oral cavity carrying out several functions (Scannapieco et al. 1995). HSAmy consists of a single polypeptide chain of 496 amino acids (Ramasubbu et al. 1996)
and exists in several isoforms in the salivary secretions.
Structurally, HSAmy consists of three domains: domain
A (residues 1–99, 170–404), domain B (residues 100–
169) and domain C (residues 405–496). The domain A
adopts a (β/α)8 -barrel structure bearing three catalytic
residues Asp197, Glu233 and Asp300. The domain B occurs as an excursion from domain A. Domain C forms
an all-β-structure and seems to be an independent do-
main with as yet unknown function (Ramasubbu et al.
1996). The molecular structure adapted by HSAmy is
similar to the other mammalian amylases including the
human pancreatic α-amylase with which it shares a very
high degree of sequence identity (∼ 97%).
HSAmy binds to numerically prominent species of
oral streptococci, such as Streptococcus gordonii, S.
crista and S. mitis (Handley et al. 1991) but not to
S. sanguis, S. oralis, S. mutans, or Actinomyces viscosus (Brown et al. 1999; Douglas 1983; Scannapieco et al.
1989, 1993). The amylase-binding bacterial species constitute a substantial proportion of the total cultivable
flora from the human teeth (Scannapieco et al. 1994;
Tseng et al. 1992). Interestingly, these bacteria appear
to colonize the mouth of animals that possess amylase
activity and may have an evolutionary advantage over
bacteria that do not possess a salivary protein-binding
property. The binding of S. gordonii G9B to HSAmy
is calcium independent, saturable and leads to a functionally irreversible complex (Scannapieco et al. 1989).
Two S. gordonii proteins, AbpA (20 kDa) and AbpB (80
kDa), mediate binding of HSAmy to the bacteria (Douglas et al. 1990; Gwynn & Douglas 1994; Rogers et al.
* Corresponding author
c 2008 Institute of Molecular Biology, Slovak Academy of Sciences
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Table 1. Mutagenic primers used in the construction of HSAmy mutants.
Mutation
Sequence
Primer
W316A
W316A
W388A
W388A
5’-CTATACTTACCTTCGCGGATGCTAGGC-3’
5’-GCCTAGCATCCGCGAAGGTAAGTATAG-3’
5’-GAACATCGAGCGCGCCAAATAAG-3’
5’-CTTATTTGGCGCGCTCGATGTTC-3’
Forward
Reverse
Forward
Reverse
rial colonization since such binding would not interfere
with the active site of the enzyme. To test whether or
not the interface between domains A and C plays a role
in bacterial binding, we generated two mutants W316A
and W388A and studied their bacterial binding ability,
hydrolysis of starch and oligosaccharide and their roles
of in acarbose-binding through crystallographic analysis.
Material and methods
Expression and purification of recombinant amylase proteins
The expression and purification of the recombinant proteins
was carried out using a Bac-To-Bac Baculovirus Expression
System as outlined previously (Mishra et al. 2002; Ragunath et al. 2002). The mutants targeting the sites 316 and
388 were generated as described earlier (Mishra et al. 2002;
Ramasubbu et al. 2004). The primers used in the study are
provided in Table 1. Approximately 5 mg of purified proteins were obtained per liter of the culture.
Fig. 1. Ribbon diagram of HSAmy showing the active site and
secondary saccharide binding sites.
1998; Scannapieco et al. 1992). The gene encoding the
20 kDa AbpA has been cloned and studied extensively
(Rogers et al. 1998; Rogers et al. 2001) and its role in
biofilm formation has been reported (Loo et al. 2000).
A recent study provided further support for the role
of AbpA in human saliva-supported biofilm formation
by S. gordonii (Rogers et al. 2001). While significant
progress has been made in studies regarding bacterial
proteins that bind to HSAmy, very little is known about
the molecular determinants of HSAmy that are essential for the interaction.
Interestingly, biochemical studies have shown the
existence of a similarity between substrate and bacterial binding sites (Scannapieco et al. 1990). Earlier crystallographic studies have shown that HSAmy possesses
four secondary saccharide-binding sites (Fig. 1) in addition to the active site (Ramasubbu et al. 2003). We
hypothesized that S. gordonii might utilize one of these
four secondary saccharide-binding sites for binding to
HSAmy. In HSAmy, two aromatic residues, W316 and
W388, occur in one of the secondary sites at the interface between the A domain and the C domain. Since
this site (site 3) is located away from the active site,
binding of bacteria at this site may be relevant to bacte-
Enzyme activity assays
Dinitrosalicylic acid assay was used for measuring the
starch-hydrolyzing activity of HSAmy and mutants at 25 ◦C
for 3 min in 20 mM phosphate buffer (pH 6.9) containing
6 mM NaCl using 1% soluble starch as substrate (Bernfeld
1955). Hydrolysis of maltoheptaose was carried out as previously described (Mishra et al. 2002; Kandra et al. 2003;
Remenyik et al. 2003; Ramasubbu et al. 2004).
Preparation of 125 I-labeled α-amylase and mutant proteins
Purified recombinant α-amylase (25 µg) was iodinated by
the chloramine-T method in 100 µL of PBS (Greenwood et
al. 1963). lodinated and not iodinated materials were separated from free iodine and reagents by gel filtration on
columns (10 mL disposable pipettes) of Bio-Gel P6DG (BioRad) equilibrated in PBS. Fractions of 1 mL were monitored
for radioactivity with a gamma counter (model 5500; Beckman). Void volume materials were used for the subsequent
studies.
In vitro interaction of 125 I-labeled α-amylase with bacteria
Streptococcus gordonii was cultured in TSB-Y and washed
and resuspended in PBS 0.1% lipid-free BSA. Cell density
was calculated at A600. Experiments were performed in
polypropylene microcentrifuge tubes precoated with 1.0%
BSA (Sigma-Aldrich) to reduce the nonspecific interaction
of the ligand with the tube walls. Reaction mixtures contained 0.1 mL of the bacterial suspension (Approximately 1
× 107 ), various amounts of 125 I-labeled protein, and buffer
added to a total volume of 200 µL. Triplicate samples were
incubated at 27 ◦C for 30 min, after which the reaction was
terminated by centrifugation followed by three 1-mL washes
with PBS. Finally, the pellets were resuspended in 200 µL
of PBS. The amount of radioactivity bound to the bacteria
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Table 2. Summary of diffraction data collection parameters and structure refinement statistics.
Parameters
W316A/acarbose complex
W388A/acarbose complex
Space group
Cell dimensions: a,b,c (Å)
Resolution range (Å)
Total/unique No. of reflections
Completeness (%): overall/last shell
Mean I/σI: overall/last shell
Multiplicity
Rmerge (%) (overall/last shell)a
Number of protein/solvent/other atoms
Number of reflections used
<B> factor (Å2 ): protein/solvent/other
R-/Rfree (%)
r.m.s. deviations: bonds (Å)/angles (◦ )
P21 21 21
52.28 × 75.05 × 135.01
50.00–2.00
195880/36537
99.3/99.3
34.2/7.6
5.4
10.1/41.8
3948/191/56
34563
33.1/37.1/37.3
18.1/21.8
0.02/1.6
P21 21 21
52.09 × 74.87 × 134.69
30.96–1.60
218198/67719
96.2/92.7
20.1/1.9
3.3
9.3/43.9
3948/201/56
64253
23.8/29.3/27.0
19.3/20.9
0.013/1.35
was measured with a gamma counter (model 5500; Beckman) using 10 µL of the sample. Negligible radioactivity
(less than 0.01%) was recovered in the control microcentrifuge tubes from reaction mixtures, which did not contain
bacteria.
Structure determination of mutant and acarbose complexes
Crystals of the mutants W316A and W388A were grown
using conditions previously described (Ramasubbu et al.
1991, 1996) using a final protein concentration of 8 mg/mL.
Diffraction quality crystals appeared over a period of one
week. The protein complexes with acarbose, were obtained
by soaking the enzyme crystals with acarbose (1 mM final concentration) in 45% (+/-)-2-Methyl-2,4-pentanediol
for 24 h and the soaked crystals were used for data collection. The crystals were mounted on loops (Hampton
Research) and flash frozen to −170 ◦C in liquid nitrogen.
Diffraction data were collected using synchrotron radiation
at the CHESS beam line A-1 at liquid nitrogen temperatures and a ADSC Quantum 4 CCD detector. A total of
360 frames were collected with an exposure time of 10–15
s and an oscillation interval of 0.5◦ to give a 99% complete
data set to 1.6 Å. Intensity data were integrated, scaled and
reduced to structure factor amplitudes using HKL2000 suite
of programs (Otwinowski & Minor 1997). The unit cell parameters were found to be isomorphous with those of the
wild type HSAmy (Ragunath et al. 2002).
Refinement of the mutant structures was carried out
using Refmac5 in the CCP4 package (CCP4 1994) wherein
cycles of rigid body refinement, positional and thermal B
factor refinements were carried out using the phase information from the native enzyme (PDB code: 1SMD). A test set
consisting of 5% of reflections was used to monitor the Rfree
behavior. Manual model rebuilding was carried out using
COOT (Emsley & Cowtan 2004). The complete polypeptide
chains of the mutants were examined with Fo-Fc and 2FoFc maps. During this process, the mutant structure clearly
showed absence of side chain density for W316 and W388 in
the individual structures. The residue at position 316 and
388 was changed to Ala to reflect the mutation and for the
remainder of the refinement these residues were treated as
such. A similar procedure was used for the refinement of the
complex structures as well.
At this stage, clear-cut continuous density corresponding to the oligosaccharide ligand was observed in
the active site region at subsites −2, −1, +1 and +2.
However, no oligosaccharide atoms were included in the
refinement until the refinement of the protein reached
convergence. The identity of the sugar moieties (either
5-hydroxymethylchonduritol or 4-amino-4,6-dideoxy-α-Dglucose or glucose) was deduced from the presence or absence of density for the hydroxyl group of the side chain at
position C5 in the ring (Ramasubbu et al. 2003). The refinement was continued by the inclusion of the sugar atoms.
Further examination of the density maps revealed no additional binding sites in the complex.
Solvent molecules were added using the arp/warp procedure (Lamzin & Wilson, 1993) in the CCP4 package. The
validity of the water molecules were assessed based on the
following criteria: (i) the presence of a peak at least 3σ in
the difference map with at least one hydrogen bond to a
protein atom (N or O) or (ii) if the located water molecule
were part of a water chain connecting protein atoms; and
(iii) the water refined with a thermal factor less than 50 Å2 .
Manual fitting was interspaced between refinements as and
when necessary. The programs PROCHECK (Laskowski et
al. 1993), CCP4 and COOT were used for model analysis of the final refined structures (Table 2). The coordinates of the mutant structures (W316A, W388A) and of the
complex structures (W316/acarbose PDB code: 3BLK and
W388/acarbose, PDB code: 3BLP) have been deposited in
the Protein Data Bank (Berman et al. 2007). Other structures used for comparison have the following PDB codes:
1SMD (HSAmy), 1MFV (HSAmy/acarbose), and 1MFU
(∆306-310HSAmy/acarbose).
Results and discussion
Enzyme hydrolytic activity of the mutants
The two mutant proteins of HSAmy were expressed using the Bac-To-Bac Baculovirus Expression system described in Material and methods and purified to study
the role of these aromatic residues in bacterial binding. The specific activities for the hydrolysis of starch
are given in Table 3. Mutation at W316 or W388 did
not affect the enzyme activity in any significant way as
evident from their comparable starch hydrolyzing activity with respect to the wild type enzyme. Figure 2
provides the HPLC profiles for the hydrolysis of maltoheptaose by wild type and mutant enzymes. As can be
seen, both mutants hydrolyze maltoheptaose and produce an action pattern similar to that of the wild type.
However, the hydrolytic ability of these two mutants is
lower compared to the wild type enzyme. Of the two
mutant enzymes, W388A appears to be less affected
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Structure-function relationships in human salivary α-amylase
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Fig. 3. Binding of [125 I]-α-amylase and mutant proteins to S.
gordonii. Increasing amounts of [125 I]-labelled proteins were incubated with 107 bacteria and the radioactivity bound to the cells
was quantified. Each point is the average of triplicate samples.
ferent as calculated by statistical analysis (p = 0.05).
Binding of 125 I-labeled mutants to S. gordonii
The bacterial binding ability of the two mutants was
unaltered with respect to the wild type (Fig. 3). This
result clearly demonstrated that the interface between
A/C domains is not involved in bacterial binding.
Fig. 2. HPLC analyses of the products of the reactions of HSAmy,
W316A and W388A enzymes with maltoheptaose. Enzymes were
at a concentration of 60 nM while the substrate used was 0.5
mM. The products were identified using standards and amylose
as described previously (Ramasubbu et al. 2003).
Table 3. Parameters for the hydrolysis of starch.
Enzyme
Specific activity against starch
HSAmy
W316A
W388A
66400 (1020)
60006 (1200)
68099 (1220)
by the mutation than the W316A enzyme. Also, the
two mutants produce less maltose than the wild type
enzyme. The differences observed in the hydrolysis of
starch as well as maltoheptaose were significantly dif-
Overall structure of the mutants
The structure of the two mutants, as with the wild type
structure of HSAmy, retains the overall structure. An
important feature of the mutant structures is that irrespective of the mutation at the A/C domain interface,
both structures in general exhibit an overall fold very
similar to the native molecule (r.m.s. deviation, 0.41 Å
for W316A/acarbose and 0.38 Å for W388A/acarbose).
Some of the salient features of the α-amylase structures
such as the chloride-binding site, the calcium-binding
site, the mobile loop containing the His305 residue are
all well-defined in the mutant complex structures. The
two tryptophan residues at positions 316 and 388 in the
mutant structures clearly show the absence of density
corresponding to the side chain (Figs 4 a,b).
Structures of the acarbose complexes
Examination of the structures of the acarbose complexes show that the saccharide binding in W316/acarbose and W388/acarbose was restricted to the active
site only. At the active site of both the mutants, clear
density was observed for 5 saccharide units (Figs 4c,d).
As with the wild type enzyme, the acarbose molecule
is modified in both the mutants involving condensation
reactions. The reactions that need to occur at the active site to generate the observed modified acarbose are
shown in Figure 5. This pathway, in essence, is the same
as outlined for the wild type (Ramasubbu et al. 2003) to
generate a hexasaccharide at the active site. However,
only a pentasaccharide was modeled due to the absence
of well-defined electron density for the HMC monomer
at the non-reducing end. Unlike the wild type/acarbose
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C. Ragunath et al.
Fig. 4. Drawing of the 2Fo-Fc difference density maps. (a) W316A and (b) W388A. The density contours are at 1.0 σ. The absence
of density for the aromatic moiety of the tryptophan side chain is evident for both mutants. The final refined coordinates of the
corresponding protein residues are overlaid and shown as thick black lines. (c) W316A/acarbose and (d) W388A/acarbose. The
density for the bound oligosaccharide, contoured at 1 σ, is shown. Note that there are only five carbohydrate moieties that could be
fitted. The identity of the residues were determined using the method described earlier (Ramasubbu et al. 2003). The final refined
coordinates of the corresponding protein or oligosaccharide residues are overlaid and are shown as thick black lines.
structure, wherein two other sites were populated by an
acarbose molecule, in the mutant/acarbose complexes
these secondary sites were not populated. Mutation of
either W316 or W388, clearly results in the inability
of this site to bind an acarbose molecule as observed
in the wild type. Interestingly, the third site involvUnauthenticated
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Structure-function relationships in human salivary α-amylase
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References
Fig. 5. A schematic showing formation of the observed oligosaccharides in the enzyme/acarbose complex involving a possible
bimolecular mechanism in the crystals of HSAmy/acarbose and
the mutant/acarbose complexes. Hmc = valienamine unit represented by a crossed square; Agl = 4-amino-4,6-dideoxy α-Dglucose represented by a square; and Glc = α-D-glucose represented by a circle. The slashed circle represents the reducing end.
Hmc-Agl is commonly known as the acarviosine moiety. The initial step is the hydrolysis of acarbose to generate a trisaccharide
and glucose. The HSAmy enzymes generate a hexasaccharide by
the condensation of two units of the trisaccharide. After condensation, the newly formed oligosaccharide must shift towards the
non-reducing end to attain the final bound state.
ing residues Y276 and W284, were also devoid of any
bound acarbose molecule in sharp contrast to the wild
type/acarbose structure. No clear perturbation of the
structure was observed around these secondary sites. It
is unclear how the absence of saccharide-binding ability at these sites could affect the enzymatic activity as
described above. One possibility could be that binding at these secondary sites, especially at Y276/W284
and/or W316/W388 could alter the water channel that
has been suggested to be important for the enzyme activity.
Conclusions
We have generated two mutants of HSAmy targeting
the aromatic residues at the interface between A and C
domains, which act as binding platforms for saccharide
binding. Our studies show the mutation did not affect
the overall conformation of HSAmy. Interestingly, these
residues only affect the oligosaccharide hydrolysis and
not starch hydrolysis. The condensation activity as observed in the crystals of HSAmy appears to be intact
in the mutant crystals as well. Finally, the inability of
these two mutants to weaken the ability of bacterial
binding suggests that these sites/residues are not involved in bacterial binding.
Acknowledgements
This project was supported by the USPHS Grant DE12585
(NR).
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Received January 14, 2008
Accepted April 15, 2008
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