Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology.

Ste24, an integral membrane protein zinc metalloprotease, is found in every kingdom of eukaryotes. It was discovered approximately 20 years ago by yeast genetic screens identifying it as a factor responsible for processing the yeast mating a-factor pheromone. In animals, Ste24 processes prelamin A, a component of the nuclear lamina; mutations in the human ortholog of Ste24 diminish its activity, giving rise to genetic diseases of accelerated aging (progerias).

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders.

Phosphoramidon inhibits the integral membrane protein zinc metalloprotease ZMPSTE24.

The integral membrane protein zinc metalloprotease ZMPSTE24 possesses a completely novel structure, comprising seven long kinked transmembrane helices that encircle a voluminous 14 000 Å cavity within the membrane. Functionally conserved soluble zinc metalloprotease residues are contained within this cavity. As part of an effort to understand the structural and functional relationships between ZMPSTE24 and soluble zinc metalloproteases, the inhibition of ZMPSTE24 by phosphoramidon [N-(α-rhamnopyranosyl-oxyhydroxyphosphinyl)-Leu-Trp], a transition-state analog and competitive inhibitor of multiple soluble zinc metalloproteases, especially gluzincins, has been characterized functionally and structurally.

The role of OleA His285 in orchestration of long-chain acyl-coenzyme A substrates.

Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a β-keto acid.

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis.

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, encodes the enzymes necessary to produce -olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching.

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis.

Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from in and purification over His and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged.

Interfacial Au nanoparticle decoration of a disulfide modified G-wire.

Background: The guanine-rich oligonucleotide (GRO), dGGGGTTGGGG (GTG), has the capacity to form a linear supramolecular polymer known as a G-wire. Individual nucleotides of the component GROs can be functionally modified to serve as site-specific attachment points in the G-wire while not interfering with its self-assembling properties. An amine linker modification to an internal thymine base of the GRO, denoted GTT*G, serves as a chemically versatile attachment site.

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue.

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin.

A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR.

Crystal structures of Xanthomonas campestris OleA reveal features that promote head-to-head condensation of two long-chain fatty acids.

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism.

Crystal structures of CO and NO adducts of MauG in complex with pre-methylamine dehydrogenase: implications for the mechanism of dioxygen activation.

MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex.

Cloning, purification, crystallization and preliminary X-ray diffraction of the OleC protein from Stenotrophomonas maltophilia involved in head-to-head hydrocarbon biosynthesis.

OleC, a biosynthetic enzyme involved in microbial hydrocarbon biosynthesis, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.4 A resolution.

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO(2) (-)) to Cl(-) and O(2). The ability of Cld to promote O(2) formation from ClO(2) (-) is unusual. Heme enzymes generally utilize ClO(2) (-) as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate.

Crystallization and preliminary X-ray diffraction of chlorite dismutase from Dechloromonas aromatica RCB.

Chlorite dismutase from Dechloromonas aromatica RCB, a novel b-type hemoprotein that catalyzes O-O bond formation, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.0 A resolution.