Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis

Abstract

X-ray structures of two enzymes in the sterol͞isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain ␣͞␤ protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an ␣͞␤ metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.H igh-throughput genome sequencing has dramatically expanded our knowledge of the proteins of the natural world. Next steps in understanding these macromolecules will involve studies of biochemical and biological function, depending critically on three-dimensional structural information. Nascent structural genomics efforts are aimed at developing experimental and computational pipelines for studying protein structure and integrating their results into the mainstream of biomedical research (1).

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

“…A KpnI-SstI fragment from pRC.V8 was transferred into pQE30 to make pH6.MA. The Gag.3h (53) sequence was PCRamplified using a variant of pRC.V8 that differed from the published pAT.V8 (44) sequence [T1327 to C, resulting in a valine to alanine substitution that does not affect infectivity, RNA packaging, or budding (8)] and inserted into pET-28TEV (54). The RSV minimal packaging signal μψ (23) was amplified from pGEM7Zf + RSV − LTR.MA (43) and transferred into pGEM7Zf + (Promega).…”

Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl- d -erythritol kinase

“…The GHMP family now also includes mevalonate 5-diphosphate decarboxylase (42) and archaeal shikimate kinase (43). Crystal structures are available for several GHMP kinases (40,42,(44)(45)(46)(47) although only HSK (38) has provided experimental details for a ternary complex. In…”

Molecular cloning, characterization and heterologous expression in Saccharomyces cerevisiae of a mevalonate diphosphate decarboxylase cDNA from Ginkgo biloba

“…Furthermore, the putative ATP binding motif (P loop) was predicted to be the same in GbMVD as in other species. Homology-based structural modelling resulted in the 3D model of GbMVD, which was similar to the crystal structure of yeast MVD (Bonanno et al 2001). The phylogenetic analysis demonstrated that GbMVD belonged to plant MVD group and that it was a more ancient MVD than other plant MVDs.…”

“…2, the a-helices were separated by three antiparallel b-sheets. It was also obvious that the conserved segments (PTAAGLASSA) in the alignments resembled P loops on the surface of a concavity, which were predicted to be responsible for ATP binding as reported in other enzymes (Bonanno et al 2001, Bork et al 1993. Because the function and structure of ScMVD had been elucidated, and both of the comparative 3D models showed high similarity, it could be speculated that GbMVD might have the same function as that of S. cerevisiae from the 3D model comparison.…”

“…For better characterization of the deduced GbMVD protein, a comparative modelling of 3D model of GbMVD was performed at http://www.expasy.org/ using SWISS-MODEL (Schwede et al 2003), and the template for modelling was the 3D structure of MVD protein from S. cerevisiae (PDB No.1FI4), the crystal structure of which had been elucidated by Bonanno et al (2001 ; Fig. 2).…”