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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. Ste24 possesses a unique "α‐barrel" structure consisting of seven transmembrane (TM) α‐helices encircling a large intramembranous cavity (~14 000 Å3). The catalytic zinc, coordinated via a HExxH...E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long‐studied class of soluble ZMPs, and as a novel cavity‐containing integral membrane protein protease has been minimally explored to date. Informed by homology to well‐characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc‐containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α‐barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α‐barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family. [ABSTRACT FROM AUTHOR]

The integral membrane protein zinc metalloprotease ZMPSTE24 possesses a completely novel structure, comprising seven long kinked transmembrane helices that encircle a voluminous 14 000 Å3 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 functional results, the determination of preliminary IC50 values by the use of an intramolecular quenched‐fluorescence fluorogenic peptide assay, indicate that phosphoramidon inhibits ZMPSTE24 in a manner consistent with competitive inhibition. The structural results, a 3.85 Å resolution X‐ray crystal structure of a ZMPSTE24–phosphoramidon complex, indicate that the overall binding mode observed between phosphoramidon and soluble gluzincins is conserved. Based on the structural data, a significantly lower potency than that observed for soluble gluzincins such as thermolysin and neprilysin is predicted. These results strongly suggest a close relationship between soluble gluzincins and the integral membrane protein zinc metalloprotease ZMPSTE24. [ABSTRACT FROM AUTHOR]

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. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X‐ray crystallography. Our data demonstrate that His285 is required for product formation, influences the thiolase nucleophile Cys143 and the acyl‐enzyme intermediate before and after transesterification, and orchestrates substrate coordination as a defining component of an oxyanion hole. As a consequence, His285 plays a key role in enabling a mechanistic strategy in OleA that is distinct from other thiolases. [ABSTRACT FROM AUTHOR]

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 Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 122.7, b = 202.9, c = 247.1 Å. [ABSTRACT FROM AUTHOR]

OleC, a biosynthetic enzyme involved in microbial hydrocarbon biosynthesis, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.4 Å resolution. The crystals belonged to space group P3121 or P3221, with unit-cell parameters a = b = 98.8, c = 141.0 Å. [ABSTRACT FROM AUTHOR]