Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

Microbial electrosynthesis: opportunities for microbial pure cultures.

Microbial electrosynthesis (MES) is an emerging technology that couples renewable electricity to microbial production processes. Although advances in MES performance have been driven largely by microbial mixed cultures, we see a great limitation in the diversity, and hence value, of products that can be achieved in undefined mixed cultures. By contrast, metabolic control of pure cultures and genetic engineering could greatly expand the scope of MES, and even of broader electrobiotechnology, to include targeted high-value products. To leverage this potential, we advocate for more efforts and activities to develop engineered electroactive microbes for synthesis, and we highlight the need for a standardized electrobioreactor infrastructure that allows the establishment and engineering of electrobioprocesses with these novel biocatalysts.

AdductHunter: identifying protein-metal complex adducts in mass spectra.

Mass spectrometry (MS) is an analytical technique for molecule identification that can be used for investigating protein-metal complex interactions. Once the MS data is collected, the mass spectra are usually interpreted manually to identify the adducts formed as a result of the interactions between proteins and metal-based species. However, with increasing resolution, dataset size, and species complexity, the time required to identify adducts and the error-prone nature of manual assignment have become limiting factors in MS analysis. AdductHunter is a open-source web-based analysis tool that  automates the peak identification process using constraint integer optimization to find feasible combinations of protein and fragments, and dynamic time warping to calculate the dissimilarity between the theoretical isotope pattern of a species and its experimental isotope peak distribution. Empirical evaluation on a collection of 22 unique MS datasetsshows fast and accurate identification of protein-metal complex adducts in deconvoluted mass spectra.

A comparative survey of the influence of small self-cleaving ribozymes on gene expression in human cell culture.

Self-cleaving ribozymes are versatile tools for synthetic biologists when it comes to controlling gene expression. Up to date, 12 different classes are known, and over the past decades more and more details about their structure, cleavage mechanisms and natural environments have been uncovered. However, when these motifs are applied to mammalian gene expression constructs, the outcome can often be unexpected. A variety of factors, such as surrounding sequences and positioning of the ribozyme influences the activity and hence performance of catalytic RNAs. While some information about the efficiency of individual ribozymes (each tested in specific contexts) is known, general trends obtained from standardized, comparable experiments are lacking, complicating decisions such as which ribozyme to choose and where to insert it into the target mRNA. In many cases, application-specific optimization is required, which can be very laborious. Here, we systematically compared different classes of ribozymes within the 3'-UTR of a given reporter gene. We then examined position-dependent effects of the best-performing ribozymes. Moreover, we tested additional variants of already widely used hammerhead ribozymes originating from various organisms. We were able to identify functional structures suited for aptazyme design and generated highly efficient hammerhead ribozyme variants originating from the human genome. The present dataset will aide decisions about how to apply ribozymes for affecting gene expression as well as for developing ribozyme-based switches for controlling gene expression in human cells.

Phosphonate and Thiasugar Analogues of Glucosamine-6-phosphate: Activation of the glmS Riboswitch and Antibiotic Activity.

The glmS riboswitch is a motif found in 5'-untranslated regions of bacterial mRNA that controls the synthesis of glucosamine-6-phosphate (GlcN6P), an essential building block for the bacterial cell wall, by a feedback mechanism. Activation of the glmS riboswitch by GlcN6P mimics interferes with the ability of bacteria to synthesize its cell wall. Accordingly, GlcN6P mimics acting as glmS activators are promising candidates for future antibiotic drugs that may overcome emerging bacterial resistance against established antibiotics. We describe the synthesis of a series of phosphonate mimics of GlcN6P as well as the thiasugar analogue of GlcN6P. The phosphonate mimics differ in their pKa value to answer the question of whether derivatives with a pKa matching that of GlcN6P would be efficient glmS activators. We found that all derivatives activate the riboswitch, however, less efficiently than GlcN6P. This observation can be explained by the missing hydrogen bonds in the case of phosphonates and is valuable information for the design of future GlcN6P mimics. The thiasugar analogue of GlcN6P on the other hand turned out to be a glmS riboswitch activator with the same activity as the natural metabolite GlcN6P. The nonphosphorylated thiasugar displayed antimicrobial activity against certain bacilli. Therefore, the compound is a promising lead structure for the development of future antibiotics with a potentially novel mode of action.

Crystal structure of a GCN5-related N-acetyltransferase from Lactobacillus curiae.

Members of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal ß0 strand and instead contains a C-terminal ß7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.

Siblings with Gorlin-Goltz syndrome associated with cardiac tumors: a case report and review of literature.

Primary cardiac tumors in children are very rare and may be associated with severe arrhythmias and sudden infant death syndrome. These cardiac arrhythmias vary depending on the location and size of the tumor. Sixty-four percent of children with cardiac fibroma, the second most common benign cardiac tumor in children, have ventricular arrhythmias, affecting therapeutic management and risk profile of these children. We report on two siblings with cardiac fibromas whose clinical presentations differed depending on their locations and size of the tumors. The first child, a three-year-old girl, was diagnosed with a cardiac fibroma in the left ventricle at the age of 8 months after surviving resuscitation due to ventricular fibrillation. Secondary prophylactic implantation of an ICD was performed. On propranolol, no further malignant arrhythmias have occurred to date. The seven-month-old brother was diagnosed postnatally with a cardiac tumor adjacent to the right ventricle. A few weeks after birth, the boy had refractory supraventricular tachycardia and ventricular arrhythmia that only resolved with amiodarone. In genetic testing, Gorlin-Goltz syndrome was diagnosed in both children. Conservative pharmacological therapy is a therapeutic strategy for asymptomatic patients with cardiac fibromas. The anti-arrhythmic medication depends on the location of the tumor. Implantation of an ICD should be performed in cases of malignant arrhythmias. In rare cases, there is an association between cardiac tumors and genetic syndromes, such as Gorlin-Goltz syndrome. These should always be considered when such a tumor is diagnosed.

Combatting over-specialization bias in growing chemical databases.

BACKGROUND: Predicting in advance the behavior of new chemical compounds can support the design process of new products by directing the research toward the most promising candidates and ruling out others. Such predictive models can be data-driven using Machine Learning or based on researchers' experience and depend on the collection of past results. In either case: models (or researchers) can only make reliable assumptions about compounds that are similar to what they have seen before. Therefore, consequent usage of these predictive models shapes the dataset and causes a continuous specialization shrinking the applicability domain of all trained models on this dataset in the future, and increasingly harming model-based exploration of the space. PROPOSED SOLUTION: In this paper, we propose CANCELS (CounterActiNg Compound spEciaLization biaS), a technique that helps to break the dataset specialization spiral. Aiming for a smooth distribution of the compounds in the dataset, we identify areas in the space that fall short and suggest additional experiments that help bridge the gap. Thereby, we generally improve the dataset quality in an entirely unsupervised manner and create awareness of potential flaws in the data. CANCELS does not aim to cover the entire compound space and hence retains a desirable degree of specialization to a specified research domain. RESULTS: An extensive set of experiments on the use-case of biodegradation pathway prediction not only reveals that the bias spiral can indeed be observed but also that CANCELS produces meaningful results. Additionally, we demonstrate that mitigating the observed bias is crucial as it cannot only intervene with the continuous specialization process, but also significantly improves a predictor's performance while reducing the number of required experiments. Overall, we believe that CANCELS can support researchers in their experimentation process to not only better understand their data and potential flaws, but also to grow the dataset in a sustainable way. All code is available under github.com/KatDost/Cancels .

A standalone editing protein deacylates mischarged canavanyl-tRNAArg to prevent canavanine incorporation into proteins.

Error-free translation of the genetic code into proteins is vitally important for all organisms. Therefore, it is crucial that the correct amino acids are loaded onto their corresponding tRNAs. This process is highly challenging when aminoacyl-tRNA-synthetases encounter structural analogues to the native substrate like the arginine antimetabolite canavanine. To circumvent deleterious incorporation due to tRNA mischarging, editing mechanisms have evolved. However, only for half of the tRNA synthetases, editing activity is known and only few specific standalone editing proteins have been described. Understanding the diverse mechanisms resulting in error-free protein synthesis is of great importance. Here, we report the discovery of a protein that is upregulated upon canavanine stimulation in bacteria that live associated with canavanine-producing plants. We demonstrate that it acts as standalone editing protein specifically deacylating canavanylated tRNAArg. We therefore propose canavanyl-tRNAArgdeacylase (CtdA) as systematic name. Knockout strains show severe growth defects in canavanine-containing media and incorporate high amounts of canavanine into the proteome. CtdA is frequently found under control of guanidine riboswitches, revealing a functional connection of canavanine and guanidine metabolisms. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Error-free translation is one of the most vital processes in all living organisms, but can be substantially challenged by compounds that mimic amino acids. Canavanine, or 5-oxa-arginine, is used as an antimetabolite by higher plants that is toxic due to its incorporation into proteins. We report the discovery of a standalone editing protein specifically deacylating canavanylated tRNAArg that enables the legume rhizosphere inhabitant Pseudomonas canavaninivorans to prevent canavanine mis-incorporation into its proteome. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Outcomes of extracorporeal membrane oxygenation and cardiopulmonary bypass in children after drowning-related resuscitation.

Drowning is one of the leading causes of accidental deaths in children worldwide. However, the use of long-term extracorporeal life support (ECLS) in this setting is not widely established, and rewarming is often achieved by short-term cardiopulmonary bypass (CPB) treatment. Thus, we sought to add our experience with this means of support as a bridge-to-recovery or to-decision. This retrospective single-center study analyzes the outcome of 11 children (median 23 months, minimum-maximum 3 months-6.5 years) who experienced drowning and subsequent cardiopulmonary resuscitation (CPR) between 2005 and 2016 and who were supported by veno-arterial extracorporeal membrane oxygenation (ECMO), CPB, or first CPB then ECMO. All but one incident took place in sweet water. Submersion time ranged between 10 and 50 minutes (median 23 minutes), water temperature between 2°C and 28°C (median 14°C), and body core temperature upon arrival in the emergency department between 20°C and 34°C (median 25°C). Nine patients underwent ongoing CPR from the scene until ECMO or CPB initiation in the operating room. The duration of ECMO or CPB before successful weaning/therapy withdrawal ranged between 2 and 322 hours (median 19 hours). A total of four patients (36%) survived neurologically mildly or not affected after 4 years of follow-up. The data indicate that survival is likely related to a shorter submersion time and lower water temperature. Resuscitation of pediatric patients after drowning has a poor outcome. However, ECMO or CPB might promote recovery in selected cases or serve as a bridge-to-decision tool.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Guanidino acid hydrolysis by the human enzyme annotated as agmatinase.

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.

Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent γ-lyase in Pseudomonadaceae and Rhizobiales.

Canavanine, the δ-oxa-analogue of arginine, is produced as one of the main nitrogen storage compounds in legume seeds and has repellent properties. Its toxicity originates from incorporation into proteins as well as arginase-mediated hydrolysis to canaline that forms stable oximes with carbonyls. So far no pathway or enzyme has been identified acting specifically on canavanine. Here we report the characterization of a novel PLP-dependent enzyme, canavanine-γ-lyase, that catalyzes the elimination of hydroxyguanidine from canavanine to subsequently yield homoserine. Homoserine-dehydrogenase, aspartate-semialdehyde-dehydrogenase and ammonium-aspartate-lyase activities are also induced for facilitating canavanine utilization. We demonstrate that this novel pathway is found in certain Pseudomonas species and the Rhizobiales symbionts of legumes. The findings broaden the diverse reactions that the versatile class of PLP-dependent enzymes is able to catalyze. Since canavanine utilization is found prominently in root-associated bacteria, it could have important implications for the establishment and maintenance of the legume rhizosphere.

Microbial Electrosynthesis of Acetate Powered by Intermittent Electricity.

Microbial electrosynthesis (MES) of acetate is a process using electrical energy to reduce CO2 to acetic acid in an integrated bioelectrochemical system. MES powered by excess renewable electricity produces carbon-neutral acetate while benefitting from inexpensive but intermittent energy sources. Interruptions in electricity supply also cause energy limitation and starvation of the microbial cells performing MES. Here, we studied the effect of intermittent electricity supply on the performance of hydrogen-mediated MES of acetate. Thermoanaerobacter kivui produced acetic acid for more than 4 months from intermittent electricity supplied in 12 h on-off cycles in a semicontinuously-fed MES system. After current interruptions, hydrogen utilization and acetate synthesis rates were severely diminished. They did not recover to the steady-state rates of continuous MES within the 12 h current-on period under most conditions. Accumulating high product (acetate) concentration exacerbated this effect and prolonged recovery. However, supply of a low background current of 1-5% of the maximum current during "off-times" reduced the impact of current interruptions on subsequent MES performance. This study presents sustained MES at a rate of up to 2 mM h-1 acetate at an average concentration of 60-90 mM by a pure thermophilic microbial culture powered by intermittent electricity. We identified product inhibition of accumulating acetic acid as a key challenge to improving the efficiency of intermittently powered MES.

Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h-1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

Pseudomonas canavaninivorans sp. nov., isolated from bean rhizosphere.

A novel canavanine-degrading bacterium, strain HB002T, was isolated from rhizosphere soil of a catch crop field collected from the island of Reichenau in Konstanz, Germany, and characterized by using polyphasic taxonomy. The facultative aerobe, rod-shaped, Gram-stain-negative bacterium was oxidase- and catalase-positive. The isolate was able to grow on canavanine as a sole carbon and nitrogen source. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed highest similarities to Pseudomonas bijieensis (L22-9T, 99.93 %), Pseudomonas brassicacearum subsp. neoaurantiaca (ATCC 49054T, 99.76 %), Pseudomonas brassicacearum subsp. brassicacearum (DBK 11T, 99.63 %), Pseudomonas thivervalensis (DSM 13194T, 99.51 %), Pseudomonas kilonensis (DSM 13647T, 99.39 %) and Pseudomonas corrugata (ATCC29736T, 99.39 %). Marker gene analysis placed the strain in the intrageneric group of Pseudomonas fluorescens, subgroup P. corrugata. In silico DNA-DNA hybridization and average nucleotide identity values were both under the recommended thresholds for species delineation. The predominant fatty acids of strain HB002T were C16 : 0, C17 : 0 cyclo ω7c and C18 : 1 ω7c. The major respiratory quinone was Q9, followed by Q8 and minor components of Q7 and Q10. Results from the phenotypic characterization showd the strain's inability to hydrolyse gelatin and to assimilate N-acetyl glucosamide and a positive enzymatic activity of acid phosphatase and naphthol-AS-BI phosphohydrolase that distinguish this strain from closely related type strains. Taken together, these results show that strain HB002T represents a novel species in the genus Pseudomonas, for which the name Pseudomonas canavaninivorans sp. nov. is proposed. The type strain is HB002T (=DSM 112525T=LMG 32336T).

Absolute characterization of high numerical aperture microscope objectives utilizing a dipole scatterer.

Measuring the aberrations of optical systems is an essential step in the fabrication of high precision optical components. Such a characterization is usually based on comparing the device under investigation with a calibrated reference object. However, when working at the cutting-edge of technology, it is increasingly difficult to provide an even better or well-known reference device. In this manuscript we present a method for the characterization of high numerical aperture microscope objectives, functioning without the need of calibrated reference optics. The technique constitutes a nanoparticle, acting as a dipole-like scatterer, that is placed in the focal volume of the microscope objective. The light that is scattered by the particle can be measured individually and serves as the reference wave in our system. Utilizing the well-characterized scattered light as nearly perfect reference wave is the main idea behind this manuscript.

Outcomes of Tricuspid Valve Detachment for Isolated Ventricular Septal Defect Closure.

Partial detachment of the septal and anterior leaflets of the tricuspid valve (TV) is a technique to visualize a perimembranous ventricular septal defect (VSD) for surgical closure in cases where the VSD is obscured by TV tissue. However, TV incision bears the risk of causing relevant postoperative TV regurgitation and higher degree atrioventricular (AV) block. A total of 40 patients were identified retrospectively in our institution who underwent isolated VSD closure between January 2013 and August 2015. Visualization of the VSD was achieved in 20 patients without and in 20 patients with additional partial detachment of the TV. The mean age of patients with partial tricuspid valve detachment (TVD) was 0.7 ± 0.1 years compared with 1 ± 0.3 years (p = 0.22) of patients without TVD. There was no difference in cardiopulmonary bypass time between patients of both groups (123 ± 11 vs. 103 ± 5 minutes, p = 0.1). Cross-clamp time was longer if the TV was detached (69 ± 5 vs. 54 ± 4 minutes, p = 0.023). There was no perioperative mortality. Echocardiography at discharge and after 2.5 years (2 months-6 years) of follow-up showed neither a postoperative increase of tricuspid regurgitation nor any relevant residual shunt. Postoperative electrocardiograms were normal without any sign of higher degree AV block. TVD offers enhanced exposure and safe treatment of VSDs. It did not result in higher rates of TV regurgitation or relevant AV block compared with the control group.

Holistic evaluation of biodegradation pathway prediction: assessing multi-step reactions and intermediate products.

The prediction of metabolism and biotransformation pathways of xenobiotics is a highly desired tool in environmental sciences, drug discovery, and (eco)toxicology. Several systems predict single transformation steps or complete pathways as series of parallel and subsequent steps. Their performance is commonly evaluated on the level of a single transformation step. Such an approach cannot account for some specific challenges that are caused by specific properties of biotransformation experiments. That is, missing transformation products in the reference data that occur only in low concentrations, e.g. transient intermediates or higher-generation metabolites. Furthermore, some rule-based prediction systems evaluate the performance only based on the defined set of transformation rules. Therefore, the performance of these models cannot be directly compared. In this paper, we introduce a new evaluation framework that extends the evaluation of biotransformation prediction from single transformations to whole pathways, taking into account multiple generations of metabolites. We introduce a procedure to address transient intermediates and propose a weighted scoring system that acknowledges the uncertainty of higher-generation metabolites. We implemented this framework in enviPath and demonstrate its strict performance metrics on predictions of in vitro biotransformation and degradation of xenobiotics in soil. Our approach is model-agnostic and can be transferred to other prediction systems. It is also capable of revealing knowledge gaps in terms of incompletely defined sets of transformation rules.