241
The results showed that the enzyme is more efficient biocatalyst in
comparison with PGAEc as regards syntheses of semi-synthetic -
lactam antibiotics. Its potential for kinetically controlled syntheses
of semisynthetic -lactam antibiotics has been shown recently [12]
and has already been used at industrial scale by Fermenta Biotech
Ltd.
Here we present results of the research into enantioselectivity of
PGAA. First, the enantioselective resolutions of racemic mixtures of
␣/-amino acids having considerable potential for pharmaceutical
applications were determined experimentally. Then we describe a
construction of a homology model of PGAA and consequent molec-
ular docking experiments employed to understand molecular basis
of PGAA enantioselectivity. Experimentally validated model of the
enzyme was used to predict PGAA enantioselectivity towards new,
hitherto non-investigated substrates.
of 0.4 mL/min)—2 mM CuSO4 containing 5% or 2% isopropanol; for
Daicel Crownpak CR(+) column (flow rate of 1 mL/min)—aqueous
solution of HClO4 (pH 1) containing methanol (10% v/v). Reten-
tion times of enantiomers of reaction products are summarized in
Supplementary data (Table S1).
E values describing enantioselectivities of PGAEc or PGAA were
determined using Eq. (1):
ꢀ
ꢁ
ln A/A0
ꢀ
ꢁ
E =
(1)
ln B/B0
where A0 and A represent the concentrations of the faster react-
ing enantiomer at the reaction times 0 and t, resp. B0 and B denote
the concentrations of the slower reacting enantiomer at the reac-
tion times 0 and t, resp. [16]. The enantiomeric ratio E was also
determined by non-linear regression using Eq. (2) which is derived
from Sihı´’s equation describing a relationship between E, degree of
conversion (c) and the enantiomeric excess eep [16].
2. Experimental
ꢂ
ꢃ
1/ E−1
(
)
ꢀ
ꢁ
ꢀ
ꢁ−E
c = 1 −
1 + eep
×
1 − eep
(2)
Recombinant strain E. coli BL21(pKX1P1) and E. coli RE3(pKA18)
was used to prepare biomass for purification of PGAA and PGAEc
,
2.5. Molecular modeling
respectively. Fed batch cultures of the strains in a stirred bioreactor
were described earlier [13,14].
A homology model of PGAA was prepared using the sequence
of amino acids from GenBank (AAY25991). The most suitable tem-
plate for modeling was identified by PSI-BLAST search using the
ExPaSy server against sequences in the Protein Data Bank. The PGAA
query sequence was then aligned with the best template—PGAEc
enzyme (PDB-ID 1GM7, sequence identity 50%) using ClustalOmega
[17]. Identity of amino acid residues forming the active site between
the template and the query was 88%. Only two substitutions
occurred within the seventeen amino acid residues forming the
active site (Val56ß → Leu56ß and Ser149␣ → Ala149␣), and no
insertions or deletions were observed for these positions. Align-
ments were prepared with a constraint between the Ser1 residue
of the query sequence and the corresponding serine residue of the
template. The homology modeling based on alignment of PGAA and
PGAEc was performed using the Swiss-model webserver [18]. The ␣
and  chains of penicillin acylases were modelled separately by cal-
culating a number of minimized intermediate models which were
ranked by the structure quality Z-score. One model of the ␣ chain
and one of the  chain had to be chosen from this ensemble of struc-
tures, not only on the basis on the Z-score, but also by taking into
account the reciprocal positions of the two chains. The final model
was evaluated using a program Verify 3D [19] with the template
validation data used as the baseline to assess the respective models.
To facilitate reproducibility of the work, the model was deposited
to Protein Model DataBase [20] under the following access code
PM0080082. The analysis of Ramachandran plot generated by RAM-
with more than 95% of residues located in the favored region, for
detailed report from the analysis see Supplementary data (Fig. S8).
This finding was further confirmed by low Z-score of −0.29 reported
by QMEAN server [22,23] indicating that the quality of the model is
comparable to the high-resolution crystal structures of proteins of
pH 7.5 using default settings. Protonation of catalytically important
to specific reaction mechanism, i.e., enantioselective hydrolysis of
N-PhAc-␣/-amino acids [24].
2.2. Enzyme purification and hydrolytic activity assay
Ec
PGAA was purified as described by Skrob et al. [11] and PGA
ˇ
according to Kutzbach and Rauenbusch [15]. Purified PGAEc and
PGAA has the specific activity of 60 and 50 U/mg protein, respec-
tively. The activity of 1 unit (U) was defined as the amount of an
enzyme producing 1 mol of phenyl acetic acid per minute from
PenG (2% w/v) in 0.05 M sodium phosphate buffer (pH 8.0) at 37 ◦C.
2.3. Chemical synthesis of N-phenylacetyl-amino acid racemic
mixtures
Phenylacetyl chloride (0.044 mol) was added dropwise to
100 mL of NaOH solution (10%, w/v) of racemic mixture of an
amino acid (0.04 mol) kept on ice bath. The reaction mixture was
acidified to pH 2 with 6 M HCl, and N-phenylacetyl amino acid (N-
PhAc amino acid) was extracted three times with ethyl acetate and
recrystallized from the ethyl acetate solution. Purity of the product
was determined by HPLC using C-8 reverse phase column. Eluent
consisted of H2O (containing 0.1% TFA): acetonitrile in ratio 7:3. For
more details on products yields, chemical shifts in the 1H NMR and
13C NMR spectra and data from MS analyses see Supplementary
data (Figs. S1–S7).
2.4. Enantioselective hydrolysis of N-PhAc-amino acid racemic
mixtures
The reactions were carried out in a pH-stat at 30 ◦C under
continuous stirring. A water solution (25 mL) containing 0.025 M
N-PhAc-amino acid racemic mixture was incubated at pH 7.5 for
30 min. 50 U of PGAEc or PGAA were added to the reaction mixture
and the pH was maintained at 7.5 by titration (2 M NH4OH).
Concentrations of reactants were analyzed by HPLC using
Dionex P580 Pump, C-8 column and a Dionex PDA-100 detector set
at 215 nm. The mobile phase consisted of a mixture of acetonitrile:
water (containing 0.1% TFA) = 3: 7.
The enantiomeric excess of the products (eep) was determined
by HPLC using Dionex P580 Pump, Sumichiral OA 5000 column and
UV detector set at 215 nm or Daicel Crownpak CR(+) column using
the Dionex PDA-100 detector set at 200 nm. Composition of eluent
solution was as follows: for Sumichiral OA 5000 column (flow rate
AutoDock Vina plug-in for PyMol [25]. Structures of substrates
were prepared using Avogadro molecular editor [26] and ener-
getically minimized in four steps of the steepest descent using
MMFF94 force field [27]. Substrate and protein structures were
converted to AutoDock Vina compliant format by AutoDock Vina