Rachel Haimovitz

Cohesin-dockerin microarray : Diverse specificities between two complementary families of interacting protein modules

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin‐dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose‐binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM‐fused cohesins were immobilized on cellulose‐coated glass slides, to which xylanase‐fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin‐dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross‐species interaction among type‐II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.

Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.

UNEXPECTED DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT IN RACEMIC MIXTURES Dedicated to the memory of Prof. Shneior Lifson - A great liberal thinker.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 °C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.

Conversion of Lysophospholipids to Cyclic Lysophosphatidic Acid by Phospholipase D

Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine in aqueous 1% Triton X-100 solution. In situ monitoring of this reaction by P NMR revealed the formation of cyclic lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an intermediate which was hydrolyzed further by the enzyme at a functionally distinct active site to lysophosphatidic acid (lyso-PA). Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was found to be stable in aqueous neutral solutions at room temperature. It was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate which was approximately 4-fold slower than the rate of formation of cyclic lyso-PA. The addition of 5-10 mM sodium vanadate could partially inhibit the ring opening reaction and thus increase substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act as a dormant configuration of the physiologically active lyso-PA or may even possess specific activities which await verification.

Subtle differences in structural transitions between poly-L- and poly-D-amino acids of equal length in water

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.

Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.

Enhancement and inhibition of snake venom phosphodiesterase activity by lysophospholipids

Lysophospholipids are liberated during venomous action. In this study we demonstrated that lysophosphatidyl choline (LPC) of various acyl chains enhances considerably the activity of snake venom phosphodiesterase (PDE). Lysophosphatidic acid (LPA) and its cyclic form (cLPA), on the other hand, were found to inhibit this enzyme in a non‐competitive (LPA) or competitive (cLPA) manner. Both of these activities may contribute to the progression and subsidence of the poisoning profile. PDE from cellular origin was not substantially affected by any of the above lysophospholipids.

Crucial Roles of Single Residues in Binding Affinity, Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface

Background: Cellulosomal cohesin-dockerin interactions show intraspecies promiscuity but interspecies specificity. Results: A combination of computations and experiments reveals single cohesin residue mutations with dramatic effects not only on binding affinity but also on specificity and promiscuity. Conclusion: Natural interspecies specificity barriers in the cohesin-dockerin interaction are easily overcome by single mutations, indicating considerable plasticity. Significance: This study sheds light on the malleability and evolvability of a high affinity interaction. Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn37 show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.

Neuronal outgrowth and rescue induced by cyclic phosphates in PC12 cells.

A series of cyclic glycerophosphates and their deoxy analogues were tested for induction of neuronal outgrowth in PC12 cells. Under chronic presence of a cyclic phosphate PC12 cells developed distinct isles of neuronal networks which covered up to 20% of the culture area, while alpha and beta glycerophosphates (the negative control compounds) did not induce any neuronal outgrowth. Distinct isles of neuronal networks were also observed upon short term application (i.e. 2 pulses of 3 hours each at day 1 and day 4) of the tested cyclic phosphates in contrast to an analogous short term exposure to NGF which was abortive. Analysis of tyrosine phosphorylation indicated a battery of phosphorylated proteins after several minutes of application of the cyclic phosphates, among which was an ERK protein of approximately 63 kD (possibly ERK7). Nerve rescue experiments were carried out with NGF differentiated PC12 cells where NGF was replaced with either 1,2 or 1,3 cyclic propanediolphosphate (1,2 cPP and 1,3 cPP) for 7 days. A distinct dose dependent preservation of neuronal network by these compounds was observed. In the control cultures NGF deprivation resulted in massive neuronal retraction and cell death. Preliminary experiments indicated that the nerve rescue by the cyclic phosphates involves the increase in the level of CASPase 6. The above findings suggest that cyclic glycerophosphates and their analogues may bear important physiological and pharmacological implications which are currently under investigation.

Phenotypic differentiation of human breast cancer cells by 1,3 cyclic propanediol phosphate.

Breast cancer cells in their virulent undifferentiated state are characterized by lack of functional estrogen receptors (ER) and/or progesterone receptors (PR) as well as relatively low levels of other normal differentiation markers such as milk proteins and lipid droplets. To date, no method for in situ elevation of the state of differentiation of breast cancer cells has yet been proven effective in patients. We have recently shown that 1,3 cyclic propanediol phosphate (1,3 cPP), an analog of 1,3 cyclic glycerophosphate (1,3 cGP), can promote morphological, neuronal-like differentiation in pheochromocytoma-12 cells in vitro. In view of this observation, we tested the potential of 1,3 cPP to elevate the state of cellular differentiation of the human breast cancer cell lines MCF-7 (ER(+)) and HCC1954 (ER(-)), as characterized by the expression of steroid receptors, casein kinase, lipid droplet histology and signal-transduction gene profiles. In the range of 5-100 microM 1,3 cPP the in vitro expression of ER-alpha, PR and casein kinase increased by approximately 2-fold while the mRNA transcription increased by 2-6-fold. Moreover, following 9-12 days of incubation with 1,3 cPP, HCC1954 cells exhibited a significant increase in the production of lipid droplets as observed by Oil Red O staining. The in vivo effect of 1,3 PP on MCF-7 xenografted into nude mice was also determined. After 4 biweekly i.p. injections of 0.5 mg 1,3 cPP per mouse, tumors in the 1,3 cPP treated virtually did not grow at all while the tumors in the control group grew rapidly. Based on these findings, we propose that this novel differentiating compound has the potential to transform the malignant tumor phenotype into a near-normal phenotype, as well as to sensitize the tumor cells to anti-estrogen therapy via upgrading the status of steroid hormone receptors.