Conformational changes and loose packing promote E. coli Tryptophanase cold lability

<p>Abstract</p> <p>Background</p> <p>Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from <it>Escherichia coli</it>. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo <it>E. coli </it>Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.</p> <p>Results</p> <p>We studied the reversible cold lability of <it>E. coli </it>Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo <it>E. coli </it>Trpase all apo forms of Trpase dissociated into dimers already at 25°C and even further upon cooling to 2°C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9Å resolution. These apo mutants were found in an open conformation compared to the closed conformation found for <it>P. vulgaris </it>in its holo form. This conformational change is further supported by a high pressure study.</p> <p>Conclusion</p> <p>We suggest that cold lability of <it>E. coli </it>Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of <it>E. coli </it>Trpase to dimers and <it>P. vulgaris </it>Trpase to monomers.</p

Primitive selection of the fittest emerging through functional synergy in nucleopeptide networks

Many fundamental cellular and viral functions, including replication and translation, involve complex ensembles hosting synergistic activity between nucleic acids and proteins/peptides. There is ample evidence indicating that the chemical precursors of both nucleic acids and peptides could be efficiently formed in the prebiotic environment. Yet, studies on nonenzymatic replication, a central mechanism driving early chemical evolution, have focused largely on the activity of each class of these molecules separately. We show here that short nucleopeptide chimeras can replicate through autocatalytic and cross-catalytic processes, governed syn-ergistically by the hybridization of the nucleobase motifs and the assembly propensity of the peptide segments. Unequal assembly-dependent replication induces clear selectivity toward the formation of a certain species within small networks of complementary nucleopeptides. The selectivity pattern may be influenced and indeed maximized to the point of almost extinction of the weakest replicator when the system is studied far from equilibrium and manipulated through changes in the physical (flow) and chemical (template and inhibition) conditions. We postulate that similar processes may have led to the emergence of the first functional nucleic-acid-peptide assemblies prior to the origin of life. Furthermore, spontaneous formation of related replicating complexes could potentially mark the initiation point for information transfer and rapid progression in complexity within primitive environments, which would have facilitated the development of a variety of functions found in extant biological assembliesThe research was supported by the H2020 FET-Open (A.d.l.E and G.A.; CLASSY project, Grant Agreement Nº 862081), an NSF-BSF grant (GA; BSF-2015671), and the Spanish Ministry of Economy and Competitivity (A.d.l.E; MINECO: CTQ-2014-53673-P, CTQ-2017-89539-P, and EUIN2017-87022). The European COST Action CM1304 funded a Short-Term Scientific Mission of S.M.R. to BGU. A.K.B. received support from the BGU Kreitmann fellowships progra

The Ups and Downs of Mutation Frequencies during Aging Can Account for the Apert Syndrome Paternal Age Effect

Apert syndrome is almost always caused by a spontaneous mutation of paternal origin in one of two nucleotides in the fibroblast growth factor receptor 2 gene (FGFR2). The incidence of this disease increases with the age of the father (paternal age effect), and this increase is greater than what would be expected based on the greater number of germ-line divisions in older men. We use a highly sensitive PCR assay to measure the frequencies of the two causal mutations in the sperm of over 300 normal donors with a wide range of ages. The mutation frequencies increase with the age of the sperm donors, and this increase is consistent with the increase in the incidence rate. In both the sperm data and the birth data, the increase is non-monotonic. Further, after normalizing for age, the two Apert syndrome mutation frequencies are correlated within individual sperm donors. We consider a mathematical model for germ-line mutation which reproduces many of the attributes of the data. This model, with other evidence, suggests that part of the increase in both the sperm data and the birth data is due to selection for mutated premeiotic cells. It is likely that a number of other genetic diseases have similar features