Biochemical and structural studies of enzymes involved in the biosynthesis of menaquinone and calicheamicin

Abstract

Menaquinone (vitamin K2) and phylloquinone (vitamin K1) are widely distributed important natural products and play a very important role as electron carriers in respiratory system (former) or photosystem I (latter) in bacteria, algae and plants. In humans and animals, despite their essential roles in bone metabolism, tissue calcification and blood coagulation, they cannot be synthetized but acquired from food, making enzymes involved in the pathway attractive targets for new drug development. Thus it is important to understand the reaction mechanism catalyzed by these enzymes. 1, 4-Dihydroxyl-2-naphthoyl-CoA (DHNA-CoA) synthase (MenB) is the sixth enzyme in the menaquinone pathway and catalyzes a multistep Claisen condensation reaction. Previously, our group has found that bicarbonate ion is crucial to the activity of a large subset of its orthologues and proposed that either the bicarbonate ion or carboxylate side chain of one conserved aspartic acid at the equivalent position plays as the base to abstract the pro-R proton in the initial enolation step. However, there are still controversies on the proton abstraction step. In order to solve the controversies on the base and proton abstraction, we chose one DHNA-CoA synthase ortholog form (ScMenB) Synchocystis sp. PCC 6803 in the phylloquinone biosynthetic pathway. As expected from our previous classification, bicarbonate ion is also essential to the activity of ScMenB from Synchocystis sp. PCC 6803. We have also determined the crystal structure of ScMenB at 2.04 Å in which bicarbonate cofactor is bound to the active site at a position equivalent to that of the side chain carboxylate of an aspartate residue conserved among bicarbonate-insensitive type II MenBs. Bicarbonate is also found in the active site of the product complex ScMenB:Sa-CoA. These results provide a solid structural basis for the bicarbonate dependence of the enzymatic activity of type I DHNA-CoA synthases. Modeling of substrate analog OSB-NCoA into the active site of the ScMenB suggests that bicarbonate takes a position suitable for abstraction of pro-R proton, consistent with a previously reported faster exchange rate of pro-R in the reaction. In the crotonase superfamily, one typical feature is the ordering of the missing A-loop in the ligand-bound state. We have determined the crystal structures type I ScMenB from Synchocystis sp. PCC 6803 and type II MtbMenB from Mycobacterium tuberculosis in complex with product analogs 1-HNA-CoA and Sa-CoA which revealed similar structural changes including folding of the A-loop into a β-hairpin and re-orientation of the C-terminal helix. A unified induced-fit mechanism was proposed to account for these structural changes which result in closure of the active site to the bulk solution. DHNA-CoA and its analogs are strong competitive inhibitors for MenB and their strong interactions cause two significant changes of the Uv-Vis spectrum of DHNA-CoA including emergence of a new peak at 350 nm and red-shift of the 392 nm peak to 414 nm. The new peak at 350 nm could be an indicator for the ordering of the A-loop under crowded conditions. We studied the macromolecular crowding effect on the folding of the A-loop with this method. The decrease in the absorbance intensity was found to be due to the polarity effect, rather than the crowding effect that disrupts the hydrogen bonding interaction between the hydroxyl group of DHNA-CoA and MenB. MenD, a thiamine diphosphate dependent enzyme catalyzes a Stetter-like 1, 4-addition of α-ketoglutarate with isochorismate to yield 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC). We have for the first time observed a protonated α-carbanion intermediate after decarboxylation for MenD by soaking the ThDP/Mg²⁺-bound crystal with α-ketoglutarate. In the structure, the carboxylate group which takes equivalent position with an acetate ion in our EcMenD:ThDP:Mg²⁺ complex structure is interacted with two important basic residues, Arg395 and Arg413. In addition, 2.9 Å distance between C_α and 4’-amino group suggests that the α-carbanion/enamine intermediate is perhaps protonated by the 4’-amino group of the pyrimidine ring of ThDP, which is not revealed previously for the ThDP-dependent enzymes. Enediyne natural products belong to a remarkable class of polyketide natural product family. Calichemicin γ1 is one of the most potent anticancer agents. However, the biosynthesis of the enediyne core remains mysterious. We have identified CalE6 in the gene cluster of calicheamicin as a methionine γ-lyase which catalyzes the elimination of methionine to yield α-ketobutyrate, ammonia and methanethiol. The methanethiol is perhaps used for the biosynthesis of the tri-sulfide in calicheamicin. We have also determined its 3-Dimensional structure with PLP in the active site. Structural comparison indicates that it is methionine γ-lyase despite its low catalytic efficiency.

Date of Award	2014
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology