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Ittiamornkul, Kuljira (2015) BIOCHEMICAL INVESTIGATION OF EARLY ENZYME PATHWAYS IN HAPALINDOLE-TYPE ALKALOID BIOSYNTHESIS. Master's Thesis, University of Pittsburgh. (Unpublished)

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Stigonematalean cyanobacteria are producers of countless secondary metabolites with diverse structures that show a variety of bioactivities. Hapalindole-type indole alkaloids produced by Fischerella sp., and Hapalosiphon sp. exhibit pharmaceutical potentials. These hapalindole-type indole alkaloids share common molecular features including an indole core, isonitrile group, which is attached with a monoterpene unit to form a tri- or tetra cylic structure. The relative stereochemical diversity across C10-C12 is conserved in the same stigonematalean species, but differs between species. The origin of sterochemical diversity was intriguing but the biosynthesis knowledge at the molecular level was absent. The initial biosynthetic hypothesis was proposed by Moore, which suggested that the biosynthesis involved a chloronium ion or proton-catalyzed polycyclization of 3-(2’-isocyanoethanyl) indole 1 (E or Z) with β-ocimene 2 (E or Z) to provide tricyclic hapalindole core intermediate, which can be oxidized to other hapalindole-type molecules.
Our group started an effort to identify the biosynthetic gene clusters of ambiguines from F. ambigua UTEX 1903 and welwitindolinones from H. welwitschii UTEX B1830. This effort revealed common biosynthetic precursors in both pathways, including GPP and (Z)-1 that contradicted the early hypothesis by Moore. It also resulted in the identification of (Z)-1 biosynthetic genes that encode three isonitrile synthases I1, I2 and I3. This discovery contrasted the early study by Brady who has shown that IsnA/B are two enzymes responsible for biosynthesis of (E)-1, where I1/I2 are homologues of IsnA and I3 is homologue of IsnB. In addition to this discovery, it was found that I1-I3 was required for (Z)-1 production in vivo, whereas I1/I3 were suffice in vitro. This finding regarding the possible redundancy of I2 led to studies, including I2 truncation, complementation and in vitro reconstitution, to investigate the role of I2. These studies collectively demonstrated the possible role of I2 is an isomerase to convert ribose-5-phosphate to ribulose-5-phosphate.
Furthermore, promiscuity of I1-I3 were investigated by using various halogenated tryptophans as substrates by in vitro and in vivo. It can be deduced that the promiscuity of I1-I3 was limited by the size of substituted halogens but not by the position of the halogens on the indole ring.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Ittiamornkul, Kuljiraitk1@pitt.eduITK1
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairLiu, Xinyuxinyuliu@pitt.eduXINYULIU
Committee MemberWeber, Stephen Gsweber@pitt.eduSWEBER
Committee MemberIslam, KabirulKAI27@pitt.eduKAI27
Date: 6 October 2015
Date Type: Publication
Defense Date: 21 November 2014
Approval Date: 6 October 2015
Submission Date: 3 December 2014
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 81
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: cyanobacteria, hapalindole-type alkaloid, biosynthesis, indole vinyl isonitrile, truncation, complementation, stigonematalean, ribose-5p, ribulose-5p
Date Deposited: 06 Oct 2015 18:21
Last Modified: 06 Oct 2020 05:15


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