The Folding and Activity of the Extracellular Lipase of Rhizopus Oryzae Are Modulated by a Prosequence

Overview

Journal Biochem J

Specialty Biochemistry

Date 1996 Oct 15

PMID 8912667

Citations 10

Authors

H D Beer

G Wohlfahrt

R D Schmid

J E McCarthy

Affiliations

Soon will be listed here.

Abstract

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.

Citing Articles

Gene Cloning, Heterologous Expression and Biochemical Characterization of A Novel Extracellular Lipase from Rhizopus Oryzae KU45.

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Structural Basis by Which the N-Terminal Polypeptide Segment of Lipase Regulates Its Substrate Binding Affinity.

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Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

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References

Gilbert H . Molecular and cellular aspects of thiol-disulfide exchange. Adv Enzymol Relat Areas Mol Biol. 1990; 63:69-172. DOI: 10.1002/9780470123096.ch2. View

Krause G, Lundstrom J, Barea J, Pueyo de la Cuesta C, Holmgren A . Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin. J Biol Chem. 1991; 266(15):9494-500. View

Kassell B, Kay J . Zymogens of proteolytic enzymes. Science. 1973; 180(4090):1022-7. DOI: 10.1126/science.180.4090.1022. View

ANFINSEN C . Principles that govern the folding of protein chains. Science. 1973; 181(4096):223-30. DOI: 10.1126/science.181.4096.223. View

Benzonana G . Some properties of an exocellular lipase from Rhizopus arrhizus. Lipids. 1974; 9(3):166-72. DOI: 10.1007/BF02532688. View

Studier F . Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli. J Bacteriol. 1975; 124(1):307-16. PMC: 235897. DOI: 10.1128/jb.124.1.307-316.1975. View

Movva N, Nakamura K, Inouye M . Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli. J Biol Chem. 1980; 255(1):27-9. View

James M, Sielecki A . Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution. Nature. 1986; 319(6048):33-8. DOI: 10.1038/319033a0. View

Munro S, Pelham H . A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987; 48(5):899-907. DOI: 10.1016/0092-8674(87)90086-9. View

10.

Kobayashi M, Kanfer J . Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D. J Neurochem. 1987; 48(5):1597-603. DOI: 10.1111/j.1471-4159.1987.tb05707.x. View

11.

Randall L, Hardy S . Unity in function in the absence of consensus in sequence: role of leader peptides in export. Science. 1989; 243(4895):1156-9. DOI: 10.1126/science.2646712. View

12.

Zhu X, Ohta Y, Jordan F, Inouye M . Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature. 1989; 339(6224):483-4. DOI: 10.1038/339483a0. View

13.

NEURATH H . Proteolytic processing and physiological regulation. Trends Biochem Sci. 1989; 14(7):268-71. DOI: 10.1016/0968-0004(89)90061-3. View

14.

Gray A, Mason A . Requirement for activin A and transforming growth factor--beta 1 pro-regions in homodimer assembly. Science. 1990; 247(4948):1328-30. DOI: 10.1126/science.2315700. View

15.

Valls L, Winther J, Stevens T . Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids. J Cell Biol. 1990; 111(2):361-8. PMC: 2116205. DOI: 10.1083/jcb.111.2.361. View

16.

Studier F, Rosenberg A, Dunn J, Dubendorff J . Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990; 185:60-89. DOI: 10.1016/0076-6879(90)85008-c. View

17.

Brzozowski A, Derewenda U, Derewenda Z, Dodson G, Lawson D, Turkenburg J . A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature. 1991; 351(6326):491-4. DOI: 10.1038/351491a0. View

18.

Ellis R, Van Der Vies S . Molecular chaperones. Annu Rev Biochem. 1991; 60:321-47. DOI: 10.1146/annurev.bi.60.070191.001541. View

19.

Chattopadhyay J, Natarajan V, SCHMID H . Membrane-associated phospholipase D activity in rat sciatic nerve. J Neurochem. 1991; 57(4):1429-36. DOI: 10.1111/j.1471-4159.1991.tb08310.x. View

20.

Winther J, Sorensen P . Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity. Proc Natl Acad Sci U S A. 1991; 88(20):9330-4. PMC: 52708. DOI: 10.1073/pnas.88.20.9330. View