Discovery of Enzymes Responsible for an Alternate Mevalonate Pathway in Haloferax volcanii

Archaea are a distinct evolutionary domain of microorganisms that contain isoprenoids in ether linkages to glycerol rather than the ester linked fatty acids that characterize membranes of bacteria or eukaryotes. Radiolabeling experiments with acetate and mevalonate strongly implicate the mevalonate pathway in providing the isoprenoids for archaeal lipid biosynthesis. However, the enzymes responsible for mevalonate metabolism in archaea have remained either cryptic or poorly characterized. To identify the enzymes responsible for the mevalonate pathway in Haloferax volcanii, open-reading frames from the H. volcanii genome were candidate screened against bacterial and eukaryotic mevalonate pathway enzymes and overexpressed in a Haloferax host. This approach has revealed that H. volcanii encodes (HVO_2419) a 3-hydroxy-3-methylglutaryl-CoA synthase (EC 2.3.310) which is the first committed step of the mevalonate pathway. Kinetic characterization shows that H. volcanii 3-hydroxy-3-methylglutaryl-CoA synthase (HvHMGCS) exhibits substrate saturation and catalytic efficiency similar to known bacterial and eukaryotic forms of the enzyme. HvHMGCS is unique in that it does not exhibit substrate inhibition by acetoacetyl-CoA. HvHMGCS is inhibited by hymeglusin, a specific inhibitor of bacterial and eukaryotic HMGCS, with experimentally determined Ki of 570 ± 120 nM and kinact of 17 ± 3 min-1. Hymeglusin also prevents the growth of H. volcanii cells in vivo suggesting the essentiality of the enzyme and the mevalonate pathway in these microbes. H. volcanii also encodes (HVO_2762) an isopentenyl monophosphate kinase (EC 2.7.4.26) and a novel decarboxylase (HVO_1412) that has been proposed, but never demonstrated, to produce isopentenyl monophosphate. This enzyme uses phosphomevalonate and ATP as substrates while exhibiting negligible decarboxylase activity with either mevalonate or mevalonate diphosphate. Phosphomevalonate decarboxylase (PMD) exhibits an IC50 = 16 μM for 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate, reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that PMD utilizes the reaction mechanism that has been demonstrated for the classical mevalonate pathway decarboxylase. These observations mark the accomplishment of the identification of a novel phosphomevalonate decarboxylase in H. volcanii. Identification and functional characterization of these enzymes also demonstrate, for the first time, the existence of the enzymes responsible for an alternate mevalonate pathway in H. volcanii

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Molecular Biology and Biochemistry (UMKC)

Grantor

University of Missouri--Kansas City

Year dc:date.issued

2014

Author dc:creator

• VanNice, John C.

Advisor dc:contributor.advisor

• Miziorko, Henry M.

Handle dc:identifier.uri

https://hdl.handle.net/10355/43897

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/43897