A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.
A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.
Chen
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Center for Biological Resource Recovery, University of Georgia, Athens 30602-7229, USA.
Li
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Ljungdahl
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D000591:Amino Acid Isomerases / Q000096:biosynthesis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002417:Cattle; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000096:biosynthesis; D005658:Fungi / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D012417:Rumen / Q000382:microbiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014714:Vertebrates; D003313:Zea mays / Q000201:enzymology
D002352:Carrier Proteins; D017931:DNA Primers; D004706:Endodeoxyribonucleases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase