# US 7,745,108 B2 · Method for identifying terpene synthase > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,108 B2 - Document ID: US7745108B2 - Status: Granted - Kind: B2 - Application number: 10/581,975 - Filed: 2004-12-08 - Published: 2007-11-15 - Granted: 2010-06-29 - Earliest claimed: 2003-12-09 - Assignees: The Regents of the University of California - Inventors: Jack D. Newman; Neil Renninger; Vincent J. J. Martin; Jay D. Keasling; Keith Kinkead Reiling - Web page: https://patentgrep.com/patent/US7745108B2 ## CPC - C12Q 1/00: Measuring or testing processes involving enzymes, nucleic acids or microorganisms (measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34); Compositions therefor; Processes of preparing such compositions - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention provides a method of identifying an enzyme, the method generally involving contacting a sample containing an enzyme with a selected enzyme substrate, where the contacting provides for covalent binding of the substrate to an amino acid of the enzyme to form a covalently modified enzyme; and determining the amino acid sequence of at least a portion of the covalently modified enzyme, using any available peptide sequencing technology, such as tandem mass spectrometry. The present invention further provides methods of identifying a nucleic acid encoding an enzyme, the methods generally involving identifying an enzyme; and, based on the amino acid sequence of at least a portion of the enzyme, designing nucleic acid probes or primers that hybridize to the nucleic acid encoding the enzyme. ## Claims ### Claim 1 A method of identifying a terpene synthase in a biological sample comprising an enzyme, the method comprising: (a) contacting the sample with a suicide substrate that covalently modifies a terpene synthase, wherein said contacting results in covalent binding of the substrate to an amino acid of the enzyme to form a covalently modified enzyme; and (b) determining an amino acid sequence of at least a portion of the covalently modified enzyme, thereby identifying the enzyme as a terpene synthase. ### Claim 2 (depends on 1) The method of claim 1, wherein the sample is a biological sample. ### Claim 3 (depends on 1) The method of claim 1, wherein the covalently modified enzyme is isolated before said determining step. ### Claim 4 (depends on 3) The method of claim 3, wherein said suicide substrate is radiolabeled. ### Claim 5 (depends on 3) The method of claim 3, wherein the covalently modified enzyme is isolated by one-dimensional isoelectric focusing. ### Claim 6 (depends on 3) The method of claim 3, wherein the covalently modified enzyme is isolated by two-dimensional gel electrophoresis. ### Claim 7 (depends on 3) The method of claim 3, wherein the covalently modified enzyme is isolated by fast protein liquid chromatography. ### Claim 8 (depends on 3) The method of claim 3, wherein the covalently modified enzyme is isolated by size-exclusion chromatography. ### Claim 9 (depends on 1) The method of claim 1, wherein the covalently modified enzyme is proteolytically cleaved with a first proteolytic enzyme before amino acid sequencing. ### Claim 10 (depends on 9) The method of claim 9, wherein the covalently modified enzyme is proteolytically cleaved with a second proteolytic enzyme before amino acid sequencing. ### Claim 11 (depends on 1) The method of claim 1, wherein said suicide substrate is a cyclopropyl-modified polyprenyl diphosphate. ### Claim 12 (depends on 11) The method of claim 11, wherein said cyclopropyl-modified polyprenyl diphosphate is selected from the group consisting of cyclopropylidene farnesyl diphosphate, cyclopropylidene geranyl diphosphate, cyclopropylidene geranylgeranyl diphosphate, cyclopropylidene geranylfarnesyl diphosphate, cyclopropylidene hexaprenyl diphosphate, cyclopropylidene heptaprenyl diphosphate, cyclopropylidene octaprenyl diphosphate, cyclopropylidene solanesyl diphosphate, cyclopropylidene decaprenyl diphosphate, cyclopropylidene undecaprenyl diphosphate, and cyclopropylidene dehydrodolichyl diphosphate. ### Claim 13 (depends on 1) The method of claim 1, wherein said suicide substrate is a vinyl analog of a polyprenyl diphosphate. ### Claim 14 (depends on 13) The method of claim 13, wherein the vinyl analog is selected from the group consisting of 6-methylidene farnesyl diphosphate, 11-methylidene geranyl diphosphate, 16-methylidene geranylgeranyl diphosphate, 21-methylidene geranylfarnesyl diphosphate, 26-methylidene hexaprenyl diphosphate, 31-methylidene heptaprenyl diphosphate, 36-methylidene octaprenyl diphosphate, 41-methylidene solanesyl diphosphate, 46-methylidene decaprenyl diphosphate, and 51-methylidene undecaprenyl diphosphate. ### Claim 15 (depends on 1) The method of claim 1, wherein said amino acid sequence determination is by tandem mass spectrometry analysis. ### Claim 16 (depends on 1) The method of claim 1, wherein said amino acid sequence determination is by Edman degradation. ### Claim 17 (depends on 1) The method of claim 1, further comprising generating a nucleic acid having a degenerate nucleotide sequence encoding the amino acid sequence, wherein the nucleotide sequence is based on the amino acid sequence determined in step (b). ### Claim 18 (depends on 17) The method of claim 17, wherein the nucleic acid is suitable for use as a hybridization probe to detect a nucleic acid that comprises a nucleotide sequence that encodes the enzyme. ### Claim 19 (depends on 17) The method of claim 17, wherein the nucleic acid is suitable for use to initiate synthesis of a nucleic acid amplification product by a DNA polymerase.