Hydrogen activation by [NiFe]-hydrogenases

Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2 The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg(509)), which interacts with two conserved aspartate residues (Asp(118) and Asp(574)) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu(28)) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg(509) (the base)

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5′-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5′-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery

Kinetically Controlled Drug Resistance:How Penicillium Brevicompactum Survives Mycophenolic Acid

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate

Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM

Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts

Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM

Abstract Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9’s CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts

Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage

Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3′O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3′ phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3′O leaving group and is the prime candidate for the general acid that protonates the 3′O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin