PMID- 33548318
OWN - NLM
STAT- MEDLINE
DCOM- 20210722
LR  - 20210722
IS  - 1879-0003 (Electronic)
IS  - 0141-8130 (Linking)
VI  - 175
DP  - 2021 Apr 1
TI  - Cloning and characterization of steroid 5beta-reductase from the venom gland of Bufo 
      bufo gargarizans.
PG  - 67-78
LID - S0141-8130(21)00259-2 [pii]
LID - 10.1016/j.ijbiomac.2021.01.211 [doi]
AB  - Bufadienolides are the main active ingredients of Venenum Bufonis, which is a 
      widely used traditional Chinese medicine secreted from parotoid gland and skin 
      glands of Bufo bufo gargarizans. According to the transcriptome analysis, 
      "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived 
      bufadienolides biosynthesis pathway by us previously. In this pathway 
      3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroid 5beta-reductase (SRD5beta) might be 
      the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the 
      second report of our group, here we report the cloning of the full length of 
      SRD5beta cDNA of B. bufo gargarizans (Bbg-SRD5beta) from the parotoid gland of B. bufo 
      gargarizans for the first time, and site-directed mutagenesis was used to 
      explored the character of Bbg-SRD5beta. Bbg-SRD5beta had an open reading frame of 
      981 bp and encoded 326 amino acids residues. The expression conditions of the 
      recombinant Bbg-SRD5beta in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5beta was 
      optimized as induction for 10 h at 15  degrees C with 0.1 mM IPTG. With NADPH as a 
      cofactor, Bbg-SRD5beta can reduce the Delta(4,5) double bonds of progesterone to 
      generate dihydroprogesterone owithout substrate inhibition effect. The catalytic 
      rate of mutant type Bbg-SRD5beta-Y132G was 1.8 times higher than that of wild type 
      Bbg-SRD5beta. Although Bbg-SRD5beta was almost unable to reduce the progesterone to 
      dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH 
      changed significantly. Bbg-SRD5beta is the key enzymes to convert the A/B ring of 
      steroid to cis-configuration, and V309 is a key site affecting the binding 
      affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic 
      rate of Bbg-SRD5beta.
CI  - Copyright (c) 2021. Published by Elsevier B.V.
FAU - Zhang, Yanan
AU  - Zhang Y
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China.
FAU - Li, Xue
AU  - Li X
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China.
FAU - Xu, Di
AU  - Xu D
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China.
FAU - Wu, Mengyun
AU  - Wu M
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China.
FAU - Dai, Yinghui
AU  - Dai Y
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China.
FAU - Xia, Mingyu
AU  - Xia M
AD  - School of Life Science and Biological Pharmacy, Shenyang Pharmaceutical 
      University, Shenyang 110016, China. Electronic address: xmywd@vip.sina.com.
FAU - Wang, Dong
AU  - Wang D
AD  - School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 
      Benxi 117004, China. Electronic address: dongwang@syphu.edu.cn.
LA  - eng
PT  - Journal Article
DEP - 20210203
PL  - Netherlands
TA  - Int J Biol Macromol
JT  - International journal of biological macromolecules
JID - 7909578
RN  - 0 (Amphibian Venoms)
RN  - 0 (Bufanolides)
RN  - 0 (DNA, Complementary)
RN  - 0 (Steroids)
RN  - EC 1.- (Oxidoreductases)
RN  - EC 1.3.99.6 (3-oxo-5 beta-steroid delta 4-dehydrogenase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amphibian Venoms/*chemistry/metabolism
MH  - Animals
MH  - Bufanolides/chemistry/metabolism
MH  - Bufo bufo/*metabolism
MH  - Bufonidae/metabolism
MH  - Cloning, Molecular/methods
MH  - DNA, Complementary/metabolism
MH  - Open Reading Frames
MH  - Oxidoreductases/genetics/*isolation & purification/metabolism
MH  - Steroids/metabolism
OTO - NOTNLM
OT  - Bufo bufo gargarizans
OT  - Mutation
OT  - Steroid 5beta-reductase
OT  - cis-configuration
COIS- Declaration of competing interest The authors declare no conflict of interest. 
      The founding sponsors had no role in the design of the study, in the collection, 
      analyses, or interpretation of data, in the writing of the manuscript, or in the 
      decision to publish the results.
EDAT- 2021/02/07 06:00
MHDA- 2021/07/23 06:00
CRDT- 2021/02/06 20:09
PHST- 2020/08/28 00:00 [received]
PHST- 2021/01/29 00:00 [revised]
PHST- 2021/01/30 00:00 [accepted]
PHST- 2021/02/07 06:00 [pubmed]
PHST- 2021/07/23 06:00 [medline]
PHST- 2021/02/06 20:09 [entrez]
AID - S0141-8130(21)00259-2 [pii]
AID - 10.1016/j.ijbiomac.2021.01.211 [doi]
PST - ppublish
SO  - Int J Biol Macromol. 2021 Apr 1;175:67-78. doi: 10.1016/j.ijbiomac.2021.01.211. 
      Epub 2021 Feb 3.