Claus Bier

Two isomorphously substituted derivatives of the nuclease-Ca2+-thymidine 3',5'-diphosphate complex have been prepared and used in an x-ray crystallographic study of the molecular structure. In one case, 5-iododeoxyuridine 3',5'-diphosphate was used in place of thymidine 3',5'-diphosphate (a net replacement of CH3 by I), and, in the other, Ba2+ was used in place of Ca2+. Intensities of all reflections and their Friedel pairs within the 4 A sphere were measured on this inhibitor complex and its two substituted derivatives; in addition, approximately 35% of the data between 4 A and 2 A were collected, selection being made on the basis of the peak to background ratios. These data have been used to produce an electron density map of sufficient quality to allow a complete tracing of the peptide chain except for a few residues at each terminus which presumably project into solution and are too disordered to be distinguishable from solvent. Approximately 30 residues are involved in three separated sections of helix, and about 24 residues form a three-stranded section of antiparallel β-pleated sheet. Residues 44 to 53 form a loop which is loose, highly exposed to solvent, and somewhat disordered. Residues Lys-48 and Lys-49, which are selectively vulnerable to trypsin-catalyzed hydrolysis, lie at the extremum of this loop. The most significant feature of the nuclease structure is a large pocket which serves as the inhibitor binding site. With the electron density maps now available, the lining of this pocket is revealed to be predominantly neutral or hydrophobic with the exception of several residues which specifically participate in binding the calcium ion and the nucleoside diphosphate. Of the latter, the most conspicuous are Lys-84 and Tyr-85 which form hydrogen bonds to the 3'-phosphate, the guanidinium moieties of Arg-35 and Arg-87 which form hydrogen bonds to the 5'-phosphate, and the carboxylate ions of Glu-43, Asp-21, and Asp-40 which serve as ligands to the calcium ion. Although the calcium ion is directly below the 5'-phosphate, it is not close enough for direct interaction with it. It appears that the barium ion occupies a position significantly different from that of the calcium ion.

Independent 4 A electron density maps calculated for the extracellular nuclease of Staphylococcus aureus (based on data from three heavy-atom derivatives) and for a nuclease-thymidine-3',5'-diphosphate-calcium ion complex (based on a single isomorphous derivative) show about 60 per cent of the chain resolved, including 3(1/2) turns of helix. The pyrimidine ring of the inhibitor fits into a pocket in the enzyme and appears to be parallel to the ring of a tyrosyl residue. Conformational changes can be observed between the nuclease and the nuclease-inhibitor complex, but the two structures seem to be identical over most of the molecule.

We will make no attempt here to present a complete description of either the crystal structure of the complex between the staphylococcal nuclease, thymidine-3′,5′-diphosphate (pdTp) and calcium ion or of the behavior of the enzyme in solution. Rather we restrict ourselves, in the first part, to (a) preliminary consideration of some of the interactions of the amino acid side chains, and (b) a brief and speculative proposal concerning the mechanism of action for the nuclease. In the second section, we shall summarize certain solution studies of this enzyme, namely those concerning the course of denaturation-renaturation and the recombination of the various fragmented forms of the nuclease and attempt to correlate these with the structure. The chemistry and structure of the nuclease are reviewed in some detail in two chapters in The Enzymes (Anfinsen et al., 1971; Cotton and Hazen, 1971).

Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-β-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCE: Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial T7 RNA polymerase expression system based on a photocaged derivative of the inducer molecule isopropyl-β-d-thiogalactopyranoside (IPTG). We have comparatively analyzed different Escherichia coli lac promoter-regulated expression systems in batch and microfluidic single-cell cultivation. The lacY-deficient E. coli strain Tuner(DE3) harboring additional plasmid-born copies of the lacI gene exhibited a sensitive and defined response to increasing IPTG concentrations. Photocaged IPTG served as a synthetic photo-switch to convert the E. coli system into an optogenetic expression module allowing for precise and gradual light-triggering of gene expression as demonstrated at the single cell level.