Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin B6 biosynthesis. The enzyme catalyzes the phosphorylation of the 5′ alcohol groups of free form ...vitamin B6 into their 5′-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of NdeI and BamHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70°C and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 – 9.0) than those previously reported. This enzyme prefers Mg2+ and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 ± 1.35 µM), while it showed the same affinity to pyridoxal (100 ± 0.76 µM) and pyridoxamine (100 ± 1.21 µM). The Km value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 ± 1.76 µM and 19 ± 0.85 µM, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin B6.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Copper(
ii
) complexes based on guanidine derivatives have been synthesized by the addition of N,N-heterocyclic ligands, yielding four new compounds, Cu(L
1
)(bipy)Cl
2
(
1
), Cu(L
1
)(phen)Cl
2
(
2
...), Cu(L
2
)(bipy)Cl
2
(
3
) and Cu(L
2
)(phen)Cl
2
(
4
) (L
1
= amidino-
O
-methylurea, L
2
=
N
-(benzyl)-amidino-
O
-methylurea, bipy = 2,2′-bipyridine and phen = 1,10-phenanthroline), and their biological activities have been studied. All complexes were characterized by elemental analysis and various spectroscopic methods (FT-IR, mass, diffuse reflectance, UV-Vis and EPR). Their structures were proposed to be square planar (for
1
,
2
and
4
) and distorted octahedral (for
3
). Their interactions with calf thymus (CT) DNA were examined by electronic absorption titration, viscosity measurements, circular dichroism spectroscopy, DNA-melting analysis, fluorescence spectroscopy and determination of the stoichiometry. Two possible DNA-binding modes of the complexes are proposed to be non-intercalation at low complex/DNA ratios and intercalation at high complex/DNA ratios. Their nuclease activities, investigated by gel-electrophoresis and atomic-force microscopy (AFM), show that the complexes can cleave plasmid pBR322 DNA, probably through oxidative pathways. Moreover, their
in vitro
cytotoxic activities against three human tumor cell lines (small cell lung carcinoma (NCI-H187), oral cavity carcinoma (KB) and breast adenocarcinoma (MCF-7)) and their antibacterial activities toward three Gram negative bacteria (
E. coli
,
Salmonella
and
Campylobacter
) were determined. The complexes in this system exhibit a more potent anticancer effect against the NCI-H187 cell line. Complex
2
had the best inhibition efficiency, particularly against
Campylobacter
. Indeed, the biological activities of the complexes follow the trend of
2
>
4
>
1
>
3
.
DNA binding and cleavage properties of two copper(II) complexes with ligands derived from guanidine have been investigated by various techniques. It was found that both complexes have the potential ...not only to interact with DNA, but also to inhibit bacterial growth.
The two designed copper(II) complexes, Cu(L
1m)
2Cl
2 (
1) (L
1m
=
amidino-
O-methylurea) and Cu(L
2m)
2Cl
2 (
2) (L
2m
=
N-methylphenyl-amidino-
O-methylurea), have been investigated for their interaction with calf thymus DNA by utilizing the absorption titration method, viscometric studies and thermal denaturation. The cleavage reaction on pBR322 DNA has been monitored by agarose gel electrophoresis. The results suggest that the two complexes can bind to DNA by non-intercalative modes and exhibit nuclease activities in which supercoiled plasmid DNA is converted to the linear form. Complex
2, with an intrinsic binding constant (
K
b) of 1.16
×
10
5
M
−1, shows a higher binding efficiency and a better nuclease activity than complex
1, with a
K
b value of 5.67
×
10
4
M
−1. Their DNA cleavage potential can be significantly enhanced by hydrogen peroxide, indicating an oxidative cleavage process. Further examination of the antibacterial activities against
Campylobacter has revealed inhibition zones of 9.0 (for
1) and 14.5
mm (for
2), which are in agreement with their minimum inhibitory concentration (MIC) values of 1.56 and 0.78
mg
mL
−1, respectively. The substantially better reactivity of
2 results from the aromatic moieties on the side chain of the L
2m ligand which act as an additional binding site.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•Two copper(II) complexes containing guanidine derivatives and deprotonated enrofloxacin as the second ligand were synthesized and characterized by X-ray crystallography.•Non-intercalation is the ...most probable DNA binding mode of the complexes.•Both complexes display significantly greater antibacterial activities than the starting complexes.•Their in vitro anticancer activities are higher and more selective towards MCF-7 and NCI-H187 than the starting complexes.
Two new copper(II) chloride complexes from guanidine derivatives containing a deprotonated enrofloxacin (erx−) as the second ligand, CuL1(erx)Cl (1) and CuL2(erx)Cl (2), were synthesized and characterized by elemental analysis and several spectroscopic methods. Crystallographic data of the single crystal of 1 gave a monoclinic with P21/n space group of Cu(L1−H+)(erx)⋅2MeOH with a slightly distorted square-planar CuN2O2 geometry. The DNA binding studies of 1 and 2 toward calf thymus (CT) DNA by absorption titration, fluorescence, viscosity measurements and circular dichroism spectroscopy showed non-intercalative binding mode with the Kb values of 1 > 2. Their cleaving ability toward plasmid pBR322 DNA was investigated by gel electrophoresis and atomic force microscopy (AFM). From these results, an oxidative mechanism is possible to be the main cleaving pathway of 1 and 2. The antibacterial activity of 1 and 2 was then tested against three human-food poisoning bacteria (E. coli, Salmonella and Campylobacter) by disc diffusion method. Both complexes exhibit inhibitory activity against E. coli and Salmonella greater than their corresponding starting complexes, CuL1Cl22 (for 1) and CuL2Cl22 (for 2). Study on the cytotoxicity of 1 and 2 against three human cancer cell lines including oral cavity (KB), breast (MCF-7) and small cell lung (NCI-H187) cell lines was determined using Resazurin Microplate assay (REMA). Results have shown that both complexes display much higher cytotoxicity against MCF-7 than their corresponding starting complexes. The better biological activities of the complexes in this system can be probably ascribed to the presence of erx−.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin B.sub.6 biosynthesis. The enzyme catalyzes the phosphorylation of the 5' alcohol groups of free ...form vitamin B.sub.6 into their 5'-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of Ndel and betaalphamHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70degreesC and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 - 9.0) than those previously reported. This enzyme prefers Mg.sup.2+ and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 + or - 1.35 microM), while it showed the same affinity to pyridoxal (100 + or - 0.76 microM) and pyridoxamine (100 + or - 1.21 microM). The K.sub.m value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 + or - 1.76 microM and 19 + or - 0.85 microM, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin B.sub.6. Keywords: pyridoxal kinase, salvage, thermophile, vitamin B.sub.6
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides ...and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciensd-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD+ to NADP+ as a cofactor, although AKRs generally show higher affinities for the latter.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The interaction of two copper(
ii
) complexes, namely Cu(
L
1
)Cl
2
2
(
1
) and Cu(
L
2
)Cl
2
2
(
2
), where
L
1
= 1-amidino-
O
-methylurea and
L
2
=
N
-(benzyl)-amidino-
O
-methylurea, with DNA ...has been thoroughly investigated using different characterization techniques, including electronic absorption spectroscopy, viscosity measurements, fluorescence spectroscopy, circular dichroism spectroscopy, thermal denaturation, stoichiometric determination, gel electrophoresis and atomic-force microscopy. The coordination compounds exhibit DNA binding potential by non-intercalation and DNA-cleaving ability through the oxidative pathway. Indeed, both complexes display antibacterial properties (against three bacteria involved in human-food poisoning,
i.e. Salmonella
,
E
.
coli
and
Campylobacter
). Furthermore, their cytotoxicity has been tested against three cancer cell lines, which are the small cell lung carcinoma (NCI-H187), the oral cavity carcinoma (KB) and the breast adenocarcinoma (MCF-7) and it was revealed that they are more cytotoxic than cisplatin against the NCI-H187 cancer cell line.
Two copper(
ii
) complexes from the water-soluble amidino-
O
-methylureas exhibit interesting activities which are of paramount importance for biological applications.
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