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Abstract: DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid de­hydrogenases. In complex with E2 (di­hydro­lipo­amide succinyltransferase, DLST) and E3 (dihydrolipo­amide de­hydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative de­carboxyl­ation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Å resolution crystal structure of human DHTKD1 is solved in complex with the thi­amine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) de­hydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Å resolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-amino­adipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

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Abstract: Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn–Teller-distorted Cu octa­hedra. The sulfate tetra­hedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra­hedra are split and rotated, which enables additional hydrogen bonds.

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Abstract: Multicopper oxidases oxidize a vast range of aromatic substrates coupled to the reduction of molecular oxygen to water. A vast broad spectrum of applications reflects their high biotechnological importance. The crystal structure of McoA from the hyperthermophilic bacteria Aquifex aeolicus has the most tightly compact and hydrophobic core among its prokaryotic counterparts. A 29-residue long loop enriched in glycines and methionines (Met-loop) close to the active T1 Cu center is not detected in the electron density maps. Accurate prediction of loop structures remains challenging, especially for long segments with sizable conformational space. Therefore, a combination of Rosetta and molecular dynamics simulations with ensemble-based small-angle X-ray scattering analysis was used to probe the conformational landscape of the Met-loop. The results indicate a highly flexible omega-loop, which is nevertheless not random but preferentially follows open-to-close transitions, exposing or occluding the T1 Cu site. Loop-truncated variants maintain wild-type stability and consistently lower and higher catalytic efficiencies (kcat/Km) for organic and metal substrates, respectively. Our results suggest that the loop transient dynamic equilibrium can exert important switch-like regulatory function, defining a role for Met-rich motifs as dynamic gate-gappers. This work provides insights into the dynamics of Met-rich loops essential to understand the molecular determinants of substrate promiscuity and catalytic rates within multicopper oxidases. We anticipate that engineering the Met-loop structural dynamics will unleash important changes in enzyme function and specificity with impact on their applications.

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Abstract: The methylation of amide nitrogen atoms can improve the stability, oral availability and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harbouring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline depending on its sequence context can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.

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Abstract: In the past couple of decades, the laser-heated diamond anvil cell (combined with in situ techniques) has become an extensively used tool for studying pressure-temperature-induced evolution of various physical (and chemical) properties of materials. In this review, the general challenges associated with the use of the laser-heated diamond anvil cells are discussed together with the recent progress in the use of this tool combined with synchrotron X-ray diffraction and absorption spectroscopy.