Discovering Enzymes for Synthesis of Antibiotics Related to Kanosamine
Date
2020-05-06
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0003-3852-2405
Type
Thesis
Degree Level
Doctoral
Abstract
Kanosamine is an antibiotic that has shown inhibitory effects on the growth of fungi and some bacteria. Kanosamine has been found to be produced by Bacillus cereus UW85 and it was predicted that the kab operon contains the genes required for kanosamine biosynthesis. The gene products of the kab operon have not previously been characterized in vitro. Kanosamine residues are found in more complex molecules, including 3,3'-neotrehalosadiamine (NTD), a reported antibiotic isolated from Bacillus subtilis. However, there are no enzymes reported to catalyze the activation or other processing of kanosamine.
This thesis describes the expression, purification, and characterization of recombinant Kab enzymes catalyzing the biosynthesis of kanosamine in three steps from glucose 6-phosphate (G6P): KabC, a NAD+-dependent dehydrogenase: KabA, a pyridoxal 5'-phosphate (PLP) dependent glutamate aminotransferase and KabB, a kanosamine 6-phosphate (K6P) phosphatase. This pathway is consistent with the previously reported kanosamine biosynthesis in Bacillus subtilis. The first steady-state kinetic parameters of three Kab enzymes are reported, along with four crystal structures of KabA: the wild-type enzyme in the presence of PLP, pyridoxamine 5'-phosphate (PMP), and two ternary complexes of the enzyme with PLP and either glutarate or K6P. Addition of glutamate resulted in formation of PMP. In the presence and absence of K6P, crystal structures show the internal and external aldimines, respectively, establishing all important active-site interactions of KabA with PLP and the aminoglycoside substrate.
A complete biosynthesis pathway of NTD is also hypothesized, one that would require more reactive kanosamine species for incorporation into more complex structures, such as NDP-kanosamine, or a closely related sugar donor, formed by a nucleotidylyltransferase. A likely first step is a phosphomutase reaction of K6P to form kanosamine 1-phosphate. The B. subtilis genome was searched for putative phosphokanosamine mutase genes. Two uncharacterized phosphomutase candidates from B. subtilis were cloned, expressed, and screened for activity. HPLC assays showed that both these enzymes exhibited phosphoglucomutase activity, but no phosphokanosamine mutase activity was observed. A putative phosphoglucomutase showed higher apparent activity and was further characterized using glucose 1,6-bisphosphate as an activator, supporting a Ping-Pong kinetic mechanism.
Description
Keywords
Kanosamine, Biosynthesis, Antibiotics, Dehydrogenase, Aminotransferase, Phosphatase, kab operon, aminotransferase structure, phosphoglucomutase
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Chemistry
Program
Chemistry