Crystal structures of the human Dysferlin inner DysF domain

Background: Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region. Results: We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Angstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing. Conclusions: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease

Resuscitation-promoting factors possess a lysozyme-like domain

The novel bacterial cytokine family – resuscitation-promoting factors (Rpfs) – share a conserved domain of uncharacterized function. Predicting the structure of this domain suggests that Rpfs possess a lysozyme-like domain. The model highlights the good conservation of residues involved in catalysis and substrate binding. A lysozyme-like function makes sense for this domain in the light of experimental characterization of the biological function of Rpfs

Letter to the Editor: 1H, 15N, and 13C chemical shift assignments of the resuscitation promoting factor domain of Rv1009 from Mycobacterium tuberculosis

International audienceNo abstract availabl

Mutation studies of the Gene Encoding YuiC, a Stationary Phase Survival Protein in Bacillus subtilis

Aims: YuiC is a stationary phase survival (Sps) protein from the Firmicute Bacillus subtilis that possesses muralytic activity to cleave bacterial cell-wall peptidoglycan. It has a small lytic transglycosylase (MltA) fold analogous to the resuscitation promoting factors (Rpfs) of Actinobacteria which have a hybrid of a mini lysozyme and soluble lytic transglycosylase (Slt35/70) fold. The present study aimed at identifying key residues of YuiC/Sps that are catalytically active and studying the effect of B. subtilis cell growth upon sps/yuiC deletion. Methodology & Results: Four forms of mutated yuiC were created through Site-directed, Ligase-Independent Mutagenesis Polymerase Chain Reaction (SLIM PCR) that include the substitutions of D129A, D151A, D162A and K102A. These individual mutated yuiC genes were cloned and expressed in the Escherichia coli BL21 (DE3) expression system and subsequently purified to homogeneity using affinity, cation exchange and size exclusion chromatography. The D129A variant was shown to be insoluble, indicating its role in maintaining the right protein folding of YuiC. The remaining three variants resulted in soluble proteins but were inactive on zymograms indicating that they may be responsible for catalysis. B. subtilis cells harbouring individual sps genes (yuiC, yabE, yocH and yorM) knocked out showed stationary phase defects and altered colony morphologies compared to the wild type. Conclusion: This study has identified the key residues involved in catalysis of YuiC, which are the D151, D162 and K102. These are conserved in Sps domains. The catalytic mechanism of YuiC is similar to the mechanism reported for Neisseria gonorrhoeae MltA. sps/yuiC knock outs have implied that each sps/yuiC has a significant role on B. subtilis late growth stage. Significance and Impact of study: The B. subtilis YuiC/Sps model has given an insight into Sps functions in the final growth stage of the Firmicutes, which members include etiologic agents of anthrax, botulism and listeriosis. Inhibition of Sps protein may inactivate pathogen replication and facilitate entrance into a non-contagious dormant sporulation stage

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation

RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation promoting factor catalytic domain

We report the first structure of the catalytic domain of RpfC (Rv1884), one of theresuscitation-promoting factors (RPFs) from Mycobacterium tuberculosis. The structure was solved using molecular replacement, once the space group had been correctly identified as twinned P21 rather than the apparent C2221 by searching for anomalous scattering sites in P1. The structure displays a very high degree of structural conservation with the structures of the catalytic domains of RpfB (Rv1009) and RpfE (Rv2450) already published. This structural conservation highlights the importance of the versatile domain composition of the RPF family

Pictet-Spenglerases in Alkaloid Biosynthesis: future applications in Biocatalysis

Pictet-Spenglerases provide a key role in the biosynthesis of many biologically-active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or b-carboline moieties), so there is interest in the characterisation and use of these enzymes as versatile biocatalysts to synthesize analogues of the corresponding natural products for drug discovery. This review discusses all known Pictet- Spenglerase enzymes and their applications as biocatalysts. Rebecca , John M. , Nicholas H. and Helen C.

'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity

How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 å resolution

AbstractBackground: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an αβ heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA.Results Reported here is the crystal structure at 2 å resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B12-binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a (β/α)8 TIM barrel domain.Conclusion The histidine–cobalt distance is very long (2.5 å compared with 1.95–2.2 å in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal–carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain

Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst