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ISSN
1672-9145
Acta Biochim Biophys Sin
2005, 37(6): 371–378 CN 31-1940/Q
Cloning and Identification of Methionine Synthase Gene from Pichia
pastoris
Lan HUANG1, Dong-Yang LI2, Shao-Xiao WANG2*, Shi-Ming
ZHANG2&, Jun-Hui CHEN1, and Xiang-Fu WU2
1 State Key Laboratory of
Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University,
Nanjing 210093, China;
2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Abstract Methionine synthase (MS)
is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share
no homology and differ in their catalytic model. Based on the conserved
sequences of metE genes from different organisms, a segment of the metE
gene was first cloned from Pichia pastoris genomic DNA by PCR, and
its 5‘ and 3‘ regions were further cloned by 5‘– and 3‘-rapid
amplification of cDNA ends (RACE), respectively. The assembled sequence reveals
an open reading frame encoding a polypeptide of 768 residues, and the deduced
product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two
domains common to MetEs. The active site is located in the C-terminal domain,
in which the residues involved in the interaction of zinc with substrates are
conserved. Homologous expression of PpMetE in P. pastoris was achieved,
and the heterologous expression of PpMetE in the S. cerevisiae strain
XJB3-1D that is MetE-defective restored the growth of the mutant on
methionine-free minimal media. The gene sequence has been submitted to
GenBank/EMBL/DDBJ under accession No. AY601648.
Key words methionine synthase; Pichia
pastoris; rapid amplification of cDNA end (RACE); functional
complementation
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Received: January 9, 2005 Accepted: March 31, 2005
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30200002)
& Present address: Weis Center for Research, Geisinger Health System,
Danville, PA 17822, USA
*Corresponding author: Tel,
86-21-54921085; Fax, 86-21-54921083; E-mail, [email protected]