E.K. Pathan, et al.
B
B
A-GeneralSubjects1864(2020)129696
NADP-GDH
NAD-GDH
followed by gel filtration chromatography. The purification protocols
are described in detail in the Supplementary information (Appendix A).
2-Ketoglutarate + NAD(P)H +NH4
L-Glutamate + NAD(P)+ + H2O
+
2.3. Phosphorylation, dephosphorylation and re-phosphorylation of NADP-
GDHs
Scheme 1. The reversible reaction catalyzed by glutamate dehydrogenases.
[8]. Recently, we observed that isoenzymes of NADP-GDH were coded
by two separate genes in B. poitrasii. BpNADPGDH I gene was found to
code for Y-form specific while the BpNADPGDH II gene for H-form
specific isoenzyme in B. poitrasii. These genes showed the cause-effect
relationship with morphological (YeH) outcomes in B. poitrasii [9].
Further, Han et al. [5] also reported that the deletion of NADP-GDH
gene affected the YeH transition in human pathogenic Candida albicans
strain. Since NADP-GDHs were found to be the regulators of morpho-
logical transition, insights into their properties will be useful in defining
their possible use as targets for new antifungal drugs.
Therefore, in the present investigations, purification and char-
acterization of NADP-GDH isoenzymes of B. poitrasii by heterologous
expression in E. coli BL-21 strain were carried out. Further, the che-
mically synthesized dimethyl esters of isophthalic acid (DMIP, struc-
tural analogs of L-glutamate) and its derivatives were screened for in-
hibition of NADP-GDH activity in vitro as well as YeH transition in B.
poitrasii, and a human pathogen Candida albicans in vivo. Furthermore,
the antifungal potential of lead molecules against human pathogenic
yeast and filamentous fungi was also evaluated.
cAMP-dependent phosphorylation, dephosphorylation, and re-
phosphorylation of purified NADP-GDHs was performed as described
earlier [7,11]. The Ca2+-Calmodulin (CaM) dependent phosphorylation
of purified NADP-GDHs was performed according to Kameshita et al.
2.4. Polyacrylamide gel electrophoresis
The polyacrylamide gel electrophoresis was done in 7.5% (w/v)
polyacrylamide gel slab at pH 8.3 as described by Laemmli [13]. The
gel was stained with Coomassie Brilliant Blue R-250 and destained
using methanol/acetic acid/water (3:1:6 by volume).
2.5. Assay of glutamate dehydrogenase (NADP-GDH, E.C. 1.4.1.4)
The NADP-GDH enzyme assay was performed at the ambient tem-
perature. The NADP-GDH activity was measured by following the
change in absorbance at 340 nm [6]. Reductive amination was mon-
itored with an appropriate amount of enzyme protein in 1 mL reaction
mixture containing 100 mM Tris/HCl, pH 8.0; 150 mM NH4Cl and
30 mM 2-ketoglutarate. The reaction was initiated by the addition of
0.125 mM NADPH. The reaction in which 2-ketoglutarate omitted was
served as a control for each reaction. One unit of enzyme activity was
defined as the amount of enzyme required to oxidize/reduce one μmol
NADPH per min per mg of protein.
2. Material and methods
2.1. Organisms and growth conditions
Benjaminiella poitrasii NCIM 1240 (National Collection of Industrial
Microorganism, Pune) was maintained and sub-cultured weekly on YPG
agar (0.3% yeast extract; 0.5% peptone; 1% glucose and 1.5% agar)
slants at 28 °C. The asexual sporangiospores obtained from 7 d grown B.
poitrasii were used for inoculum development. To obtain yeast cells,
8 × 106 sporangiospores/50 mL were inoculated in YP medium con-
taining 1% glucose and incubated at 180 rpm and 37 °C for 24 h. The
hyphal cells were obtained by inoculating 8 × 106 sporangiospores/
50 mL of YP medium and incubated at 28 °C, 180 rpm for 24 h.
The other fungal strains used to test antifungal compounds in the
present study viz., Candida albicans ATCC10231, Candida albicans
ATCC24433, Candida albicans strain 1, Candida tropicalis ATCC750,
Candida glabrata NCYC388, and Cryptococcus neoformans ATCC34664
were maintained YPG agar slants, whereas Aspergillus niger ATCC10578
was maintained on potato dextrose agar (PDA, Himedia, India) slant.
All the cultures were sub-cultured on fresh slants every month. For long
term storage, the cultures grown in YPG media were kept in 30% gly-
cerol at −80 °C.
2.6. Protein estimation
Protein was estimated according to Lowry et al. (1951) method
using crystalline bovine serum albumin as a standard.
2.7. Design and synthesis of DMIP compounds
All the chemical synthesis reactions were carried out in anhydrous
solvents under Argon atmosphere using analytical reagents and solvents
in oven-dried glassware. For the synthesis of DMIP 1 and 2, sulphuric
acid was added to the stirring solution of isophthalic acid in the cor-
responding catalytic amount and mixture was refluxed at 70 °C for 4 h.
The solvent was evaporated under reduced pressure and the mixture
was dissolved in ethyl acetate, washed with brine (NaCl) and dried over
Na2SO4. The organic phase was further concentrated under reduced
pressure and the crude residue was purified over silica gel (ethyl acetate
and pet ether as eluent) to obtain DMIP 1 and DMIP 2.
For the synthesis of DMIP 3–8, the triethylamine and corresponding
amine in DMF were stirred at RT for 30 min. To this, isophthaloyl
dichloride was added dropwise for 20 min under constant stirring at RT
for 4 h. The reaction mixture was diluted with 30 mL of water and
extracted with ethyl acetate (30 mL × 3). The organic layer combined
and was dried over Na2SO4 and the solvent was removed under reduced
pressure. The crude mixtures were purified over silica gel (ethyl acetate
and pet ether as eluent) to obtain DMIP 3 to 8. All the DMIP compounds
were purified by column chromatography using spectrochem silica gel
(60–120, 100–200, 230–400 mesh). The 1H and 13C NMR spectroscopy
measurements were carried out on Bruker AC 200 MHz or Bruker DRX
400 and Bruker DRX 500 MHz spectrometers, using TMS as an internal
standard. 1H and 13C NMR chemical shifts were reported in ppm
downfield from Chloroform‑d (δ = 7.25), DMSO‑d6 (δ = 2.5) or TMS
and coupling constants (J) were reported in Hertz (Hz). Mass spectro-
scopy was carried out on PI QStar Pulsar (Hybrid Quadrupole-TOF LC/
MS/MS) and 4800 plus MALDI TOF/TOF (Applied Biosystems, USA).
The transformed Escherichia coli BL-21 strain was stored at −80 °C
in the form of glycerol stock and revived by spreading on Luria Bertani
(LB) agar medium supplemented with ampicillin (100 μg/mL) [10]. The
plates were incubated at 37 °C until colonies were observed (16–20 h).
The transformed bacterial cultures were maintained on LB agar slants
containing ampicillin (100 μg/mL) for routine use. For permanent
storage, bacterial cells were stored at −80 °C in 30% (v/v) glycerol.
2.2. Purification of NADP-GDH isoenzymes of B. poitrasii by heterologous
expression in E. coli BL-21
The deduced amino acid sequences of BpNADPGDH
I and
BpNADPGDH II were deposited in NCBI GenBank under the accession
BpNADPGDH II showed a continuous open reading frame of 455 and
456 amino acids that were cloned under the IPTG inducible promoter in
pET6XHN vector and the respective proteins were purified after het-
erologous expression in E. coli BL 21 (DE3) cells by Ni-NTA affinity
2