PMID- 30170058
OWN - NLM
STAT- MEDLINE
DCOM- 20190104
LR  - 20190104
IS  - 1879-0003 (Electronic)
IS  - 0141-8130 (Linking)
VI  - 120
IP  - Pt A
DP  - 2018 Dec
TI  - Cloning, prokaryotic expression and function of the Bufo bufo gargarizans 
      3beta-hydroxysteroid dehydrogenase (3betaHSD) gene.
PG  - 673-682
LID - S0141-8130(18)32441-3 [pii]
LID - 10.1016/j.ijbiomac.2018.08.165 [doi]
AB  - Bufadienolides, one kind of steroids, are the major active component secreted by 
      ear-side gland of Bufo species. Preliminary studies on high-throughput 
      transcriptome sequencing about B. bufo gargarizans showed that the expression of 
      3beta-Hydroxysteroid dehydrogenase (3betaHSD) in ear-side gland was nearly 20 times 
      higher than that in liver. The enzyme 3betaHSD is an essential step in the 
      biosynthesis of steroid such as progesterone, estrogens and androgens in 
      steroidogenic tissues. Accordingly, 3betaHSD is probably an important enzyme 
      involved in the biosynthesis of bufadienolides. In this study, Bbg-3betaHSD cDNA was 
      cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering 
      techniques were used to construct a recombinant prokaryotic fusion expression 
      plasmid pCOLD-Bbg3betaHSD which was introduced into E. coli BL21 for prokaryotic 
      expression. Bbg-3betaHSD has an open reading frame (ORF) of 1134 bp and encodes 377 
      amino acid residues. The speculated protein molecular weight is 42.8 kDa and its 
      theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis 
      showed that Bbg-3betaHSD was highly homologous to the 3betaHSD protein of other 
      species. Phylogenetic tree showed the highest similarity between Bbg-3betaHSD and 
      3betaHSD from Rana rugosa. The optimized expression of recombinant Bbg-3betaHSD were 
      achieved by inducing with 0.1 mmol L(-1) IPTG at 15  degrees C for 20 h. Enzymatic 
      activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 
      3beta-oxidized by Bbg-3betaHSD when NAD(+) was used as the coenzyme. Enzymatic 
      properties showed that the optimum reaction temperature of recombinant Bbg-3betaHSD 
      was 40  degrees C, the optimum pH was 8.5, and the optimum coenzyme concentration was 
      1.5 mmol L(-1).
CI  - Copyright (c) 2018. Published by Elsevier B.V.
FAU - Xu, Di
AU  - Xu D
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Wu, Mengyun
AU  - Wu M
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Li, Xue
AU  - Li X
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Xia, Mingyu
AU  - Xia M
AD  - School of Life science and Biopharmaceutics, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Liu, Dongchun
AU  - Liu D
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Dai, Yinghui
AU  - Dai Y
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Yu, Qing
AU  - Yu Q
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China.
FAU - Wu, Bin
AU  - Wu B
AD  - Shanghai Center for Drug Evaluation and Inspection, Cailun 781, Shanghai, 201203, 
      China. Electronic address: yswubin@smda.gov.cn.
FAU - Wang, Dong
AU  - Wang D
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Shenyang 110016, China. Electronic address: dongwang@syphu.edu.cn.
LA  - eng
PT  - Journal Article
DEP - 20180829
PL  - Netherlands
TA  - Int J Biol Macromol
JT  - International journal of biological macromolecules
JID - 7909578
RN  - 0 (Amphibian Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0U46U6E8UK (NAD)
RN  - 459AG36T1B (Dehydroepiandrosterone)
RN  - 73R90F7MQ8 (Pregnenolone)
RN  - EC 1.1.- (3-Hydroxysteroid Dehydrogenases)
SB  - IM
MH  - 3-Hydroxysteroid Dehydrogenases/*chemistry/genetics/metabolism
MH  - Amino Acid Sequence
MH  - Amphibian Proteins/*chemistry/genetics/metabolism
MH  - Animals
MH  - Bufo bufo/*metabolism
MH  - Cloning, Molecular
MH  - Dehydroepiandrosterone/*chemistry/metabolism
MH  - Escherichia coli/genetics/metabolism
MH  - Gene Expression
MH  - Genetic Vectors/chemistry/metabolism
MH  - Isoelectric Point
MH  - Kinetics
MH  - Molecular Weight
MH  - NAD/*chemistry/metabolism
MH  - Open Reading Frames
MH  - Phylogeny
MH  - Pregnenolone/*chemistry/metabolism
MH  - Protein Conformation, alpha-Helical
MH  - Protein Conformation, beta-Strand
MH  - Recombinant Fusion Proteins/chemistry/genetics/metabolism
MH  - Sequence Alignment
MH  - Sequence Homology, Amino Acid
MH  - Substrate Specificity
OTO - NOTNLM
OT  - 3beta-hydroxysteroid dehydrogenase
OT  - Bufo bufo gargarizans
OT  - Enzyme activity
OT  - Prokaryotic expression
EDAT- 2018/09/01 06:00
MHDA- 2019/01/05 06:00
CRDT- 2018/09/01 06:00
PHST- 2018/05/20 00:00 [received]
PHST- 2018/08/24 00:00 [revised]
PHST- 2018/08/27 00:00 [accepted]
PHST- 2018/09/01 06:00 [pubmed]
PHST- 2019/01/05 06:00 [medline]
PHST- 2018/09/01 06:00 [entrez]
AID - S0141-8130(18)32441-3 [pii]
AID - 10.1016/j.ijbiomac.2018.08.165 [doi]
PST - ppublish
SO  - Int J Biol Macromol. 2018 Dec;120(Pt A):673-682. doi: 
      10.1016/j.ijbiomac.2018.08.165. Epub 2018 Aug 29.