Moshe Goldsmith

Moshe Goldsmith
Weizmann Institute of Science | weizmann · Department of Biomolecular Sciences

PhD

About

42
Publications
3,953
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1,713
Citations

Publications

Publications (42)
Article
Full-text available
Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed a...
Article
Full-text available
Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels...
Preprint
Full-text available
Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, b-N-oxalyl-L-a,bdiaminopropionic acid (b-ODAP), which can cause a severe neurological disorder if consumed as a...
Preprint
Full-text available
Grass pea ( Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, β- N -oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder if consumed...
Article
Full-text available
Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO 2 . Several photorespiration bypasses have been previously suggested but were limited to e...
Article
Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analys...
Article
Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the o...
Article
The practical need for highly efficient enzymes presents new challenges in enzyme engineering, in particular, the need to improve catalytic turnover (kcat) or efficiency (kcat/KM) by several orders of magnitude. However, optimizing catalysis demands navigation through complex and rugged fitness landscapes, with optimization trajectories often leadi...
Article
Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau....
Article
Full-text available
The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their effic...
Data
Document S1. Figures S1–S3, Tables S1–S6, and Supplemental Experimental Procedures
Data
Data S2. Protein and DNA Sequences of Experimentally Tested Constructs, Related to Experimental Procedures
Data
Data S1. RosettaScripts, flags, and Command Lines, Related to Experimental Procedures
Article
The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonis...
Article
Full-text available
Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an...
Article
Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival ra...
Article
Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug tre...
Article
Full-text available
The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE)...
Article
Protein engineering by directed evolution relies on the use of libraries enriched with beneficial variants. Such libraries should explore large mutational diversities while avoiding high loads of deleterious mutations. Here we describe a simple protocol for incorporating synthetic oligonucleotides that encode designed, site-specific mutations by as...
Patent
Full-text available
An isolated polypeptide comprising the amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency of kcat/KM≈106-5·107 M−1min−1 for a G-type organophosphate.
Article
The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a...
Article
This review outlines the strategies we apply for directed enzyme evolution using targeted libraries, namely, libraries that diversify specific residues with predefined mutational compositions. The theoretical grounds underlining the design of such libraries are described, including the mutational load, the ratio of beneficial versus deleterious mut...
Article
The field of directed evolution has progressed to the point where it is feasible to engineer enzymes for unnatural substrates and reactions with catalytic efficiencies and regio-specificity or stereo-specificity that rival those of natural enzymes. Here, we describe the conceptual and methodological advances that have enabled this progress. We addr...
Article
A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type n...
Article
Full-text available
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze th...
Article
An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conf...
Patent
An isolated polypeptide comprising the amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency of K cat /K m ≈106 M-1min-1 for a nerve-agent substrate.
Article
Full-text available
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved P...
Article
Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentiall...
Article
Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistanc...
Article
Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we fo...
Article
Full-text available
This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants' -- i.e. cells transformed with, or infected by, two different plasmids simult...
Article
Full-text available
Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by...
Article
Full-text available
Replication through damaged sites in DNA requires in Escherichia coli the SOS stress-inducible DNA polymerase V (UmuC), which is specialized for lesion bypass. Homologs of the umuC gene were found on native conjugative plasmids, which often carry multiple antibiotic-resistant genes. MucB is a UmuC homolog present on plasmid R46, and its variant pla...

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