Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103.
BACKGROUND
The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: alpha-, beta-, gamma-, delta-, epsilon-, zeta- CAs, with beta-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of beta-CA Nce103/YNL036W.


RESULTS
We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 A resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.


CONCLUSIONS
The quaternary structure of Nce103 resembles the typical plant type beta-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.
Teng
Yan-Bin
YB
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, PR China. ybteng@mail.ustc.edu.cn
Jiang
Yong-Liang
YL
He
Yong-Xing
YX
He
Wei-Wei
WW
Lian
Fu-Ming
FM
Chen
Yuxing
Y
Zhou
Cong-Zhao
CZ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019281:Dimerization; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D015032:Zinc / Q000737:chemistry
D002256:Carbonic Anhydrases; D015032:Zinc