Molecular characterisation of adenosylhomocysteinase from Trichomonas vaginalis.
The enzyme S-adenosylhomocysteine hydrolase (SAHH) has been identified as a potential target for chemotherapy in protozoan parasites including Trichomonas vaginalis. To investigate this area of trichomonad metabolism in more detail, we have isolated and characterised a gene which encodes this activity from the WAA38 strain of this parasite. The gene was isolated by probing a Bg/II genomic mini-library with a fragment of the gene generated by thermal cycling using degenerate oligonucleotide primers. A 5.9-kb Bg/II clone was isolated and has been partially sequenced to reveal a 1458-bp open reading frame which encodes a 486-residue polypeptide (computed molecular mass of 53.4 kDa). The deduced amino acid sequence showed a high degree of sequence similarity to the hydrolases from other species, but was most similar to the enzyme from photosynthetic organisms. The trichomonal sahh gene also contains two "insertion sequences', one of which appears to be unique to this parasite while the second has previously been found only in photosynthetic organisms and in Plasmodium falciparum. Characterisation of the sahh mRNA from T. vaginalis confirmed that both of these insertion sequences (encoding 9 and 37 amino acid residues, respectively) are expressed in the protein product. The sahh mRNA is similar to those characterised from other protozoa in having a short, 12-bp untranslated 5'-leader sequence but the leader sequence does not conform well with the consensus sequence of the other mRNAs. Finally, Southern blots and sequence differences between genomic and cDNA clones indicate that there are multiple copies of the sahh gene in T. vaginalis.
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia. a.bagnara@unsw.edu.au
A S
AS
Bagnara
V E
VE
Tucker
L
L
Minotto
E R
ER
Howes
G A
GA
Ko
M R
MR
Edwards
I W
IW
Dawes
D043383:Adenosylhomocysteinase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D006867:Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D012333:RNA, Messenger; D006867:Hydrolases; D043383:Adenosylhomocysteinase