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SummaryNovP is an S-adenosyl-L-methionine-dependent O-methyltransferase that catalyses the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at the 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated co-substrate, S-adenosyl-L-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the co-substrate binding pocket, and an active centre that contains a 3-Asp putative metal-binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilise NovP for methylating a range of glycosylated compounds.

This work describes the purification and preliminary crystallographic analysis of the CBS-pair regulatory domain of the human ancient domain protein 4 (ACDP4), also known as CNNM4. ACDP proteins represent the least-studied members of the eight different types of magnesium transporters that have been identified in mammals to date. In humans the ACDP family includes four members: CNNM1-4. CNNM1 acts as a cytosolic copper chaperone and has been associated with urofacial syndrome, whereas CNNM2 and CNNM4 have been identified as magnesium transporters. Interestingly, mutations in the CNNM4 gene have clinical consequences that are limited to retinal function and biomineralization and are considered to be the cause of Jalili syndrome, which consists of autosomal recessive cone-rod dystrophy and amelogenesis imperfecta. The truncated protein was overexpressed, purified and crystallized in the orthorhombic space group C222. The crystals diffracted X-rays to 3.6 Å resolution using synchrotron radiation. Matthews volume calculations suggested the presence of two molecules in the asymmetric unit, which were likely to correspond to a CBS module of the CBS pair of CNNM4.

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-l-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 Å , = 104.97 . Native data to a maximum resolution of 1.4 Å were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

In mammals, AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit () and two regulatory subunits (and). The subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. This work describes the purification and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, an archaeal homologue of-AMPK. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for MJ1225 were collected to 2.3 Å resolution using synchrotron radiation. The crystals belonged to space group H32, with unitcell parameters a = b = 108.95, c = 148.08 Å , = = 90.00, = 120.00. Preliminary analysis of the X-ray data indicated that there was one molecule per asymmetric unit.