Characterization of his-tagged rat uroporphyrinogen III synthase wild-type and variant enzymes.
The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Urogen III synthase catalyzes a key step in the formation of urogen III, a common intermediate for tetrapyrrolic natural products. In the present study, we cloned, purified, and characterized His-tagged rat urogen III synthase. The mechanism of enzymatic reaction was studied through site-directed mutagenesis of eight highly conserved residues with functional side chains around the active site followed with activity tests. Lys10, Asp17, Glu68, Tyr97, Asn121, Lys147, and His173 have not been studied previously, which were found to be unessential for enzymatic reaction. Tyr168 was identified as an important residue for enzymatic reaction catalyzed by rat urogen III synthase. Molecular modeling suggests the hydroxyl group of Tyr168 side chain is 3.5 A away from the D ring, and is within hydrogen bond distance (1.9 A) with acetate side chain of the D ring.
Li
Nan
N
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Ma
Dik-Lung
DL
Liu
Xiaojun
X
Wu
Long
L
Chu
Xiusheng
X
Wong
Kwok-Yin
KY
Li
Ding
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015723:Gene Library; D006639:Histidine / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014576:Uroporphyrinogen III Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006639:Histidine; D014576:Uroporphyrinogen III Synthetase