Ribonucleotide Reductases: Radical Chemistry and Inhibition at the Active Site

Abstract

Ribonucleoside 5′-diphosphate reductases (RDPRs) have been studied for several decades. Increasingly sophisticated mechanisms have been proposed for the reduction of natural substrate ribonucleotides to their 2′-deoxy counterparts and for mechanism-based inactivation of RDPRs with 2′-substituted-ribonucleotides. We now discuss biomimetic reactions of model substrate and inhibitor analogues, which clarify three aspects of previously proposed mechanisms postulated to occur at the active site of RDPRs.     

Application of Oligonucleoside Methylphosphonates in the Studies on Phosphodiester Hydrolysis by Serratia Endonuclease

Abstract

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg²⁺ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.     

非核糖体肽Skyllamycin B生物合成中I型硫酯酶底物杂泛性的初步研究

[背景] Skyllamycins是一类从链霉菌中发现的具有血小板生长因子抑制和生物膜抑制作用的非核糖体肽类,其环肽环合反应是由非核糖体肽合成酶中的硫酯酶功能域催化完成。[目的] 克隆和表达Skyllamycin非核糖体肽合成酶最后一个模块中的硫酯酶（Skyxy-TE）基因,合成Skyxy-TE底物类似物,通过体外催化实验表征Skyxy-TE的底物杂泛性。[方法] 采用Ligation Independent Cloning（LIC）方法,从一株含有Skyllamycin B生物合成基因簇的链霉菌Streptomyces sp.PKU-MA01239中克隆和表达skyxy-TE,通过镍离子柱亲和层析纯化Skyxy-TE。运用固相多肽合成法合成2个底物类似物1和2,进行Skyxy-TE的体外催化实验。[结果] 通过对Skyxy-TE的表达纯化,获得了纯度较好的可溶性蛋白;通过固相多肽合成,得到了能够模拟Skyllamycin B底物类似物的化合物1和2,硫酯酶蛋白体外催化化合物1和2得到了化合物3和4,化合物3和4通过核磁共振和高分辨质谱确认为环肽。[结论] Skyllamycin B生物合成中Skyxy-TE表现出一定的底物杂泛性,可以识别底物类似物催化环化反应,该研究为将来利用化学-酶联法制备更多环肽类似物提供了依据。     

On the packing density of the unbound protein-protein interaction interface and its implications in dynamics

Background

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities.

Results

In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool.

Conclusion

A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

     

A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G

A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration. The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281 Received 07 January 2002/ Accepted in revised form 22 May 2002     

Diffusion and Catalysis Processes in Unilamellar Liposomes

Abstract

This research concerns the study of enzyme loaded unilamellar vesicles as bioreactors. The plant enzyme Ascorbic Acid Oxidase (AAO) was entrapped in unilamellar liposomes.

Kinetic study shows that the enzyme activity is largely criptic and that the enzyme in liposomes is partially protected against proteolysis. In order to describe the reactivity of such a system, a kinetic model was developed which accounts both for the enzyme location and for the substrate diffusion across the lipid membrane. For the building of the kinetic model we have used information derived from previous studies of freeze-fracture and label fracture microscopy, which have shown that the protein is located both inside the aqueous core and across the lipid membrane of the vesicles.     

Ordered chimerogenesis applied to CYP2B P450 enzymes

BackgroundStructural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.MethodsA series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.ResultsMost of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels.ConclusionsSome sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.General significanceThe result provides a new basis for determining the structural elements that control functions in complex enzymes.     

Structural basis for carbon dioxide binding by 2-ketopropyl coenzyme M oxidoreductase/carboxylase

The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been determined in a state in which CO₂ is observed providing insights into the mechanism of carboxylation. In the substrate encapsulated state of the enzyme, CO₂ is bound at the base of a narrow hydrophobic substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic to hydrophilic environment where CO₂ is located in position for attack on the carbanion of the ketopropyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively discriminates against H₂O and prevents protonation of the ketopropyl leaving group.

Structured summary

2-KPCCbinds to 2-KPCC by x-ray crystallography (View interaction)     

Evaluation of an Alkyne-containing Analogue of Farnesyl Diphosphate as a Dual Substrate for Protein-prenyltransferases

The development of tools for proteomic analysis is an active area of research. Here, we report on the synthesis of 12-propargoxyfarnesyl diphosphate (1), an alkyne-containing analogue of farnesyl diphosphate (FPP), and its enzymatic incorporation into peptide substrates by both protein-farnesyltransferase (PFTase) and protein-geranylgeranyltransferase type I (PGGTase-I). Compound 1 was prepared from farnesol in 6 steps. Kinetic analyses indicate that 1 is incorporated into cognate peptide substrates by PFTase or PGGTase at concentrations and rates comparable to those of the natural lipid substrates for these enzymes, and mass spectrometric analyses proved the structures of the prenylated peptide products. Incubation of 1 in the presence of PFTase and PGGTase peptide substrates, and the cognate transferases, results in the simultaneous prenylation of both peptides emphasizing the dual substrate nature of 1. Thus, because 1 is a substrate for both enzymes, it can be used to introduce alkyne functionality into proteins that are normally either farnesylated or geranylgeranylated. This approach should be useful for a broad range of applications ranging from selective protein labeling to proteomic analysis. This paper is dedicated to the memory of Bruce Merrifield (1921–2006) for his pioneering development of solid-phase peptide synthesis, which has made possible myriad advances in chemical biology. For the present study, we used SPPS to prepare protein fragments that incorporate spectroscopic probes to reveal critical features in enzyme substrate recognition that have implications for human health.     

3D-QSAR with CoMFA Model of Prolylendopeptidase Substrates

Abstract

The prolylendopeptidase (PEP) is the proteolytic enzyme, which plays an essential role in the regulation of some processes in central nervous system, such as memory, learning and behavior. It was shown that PEP activity changes at different diseases, like Parkinsons or Alzheimer's diseases, and some PEP inhibitors are used in therapy. At present time the discovery of new types of PEP inhibitors are the actual task.

In this study the structure of PEP active site was analyzed by 3D-QSAR with CoMFA methods using of 12 PEP substrates. The designed pharmacophore model assumes that substrates interact with PEP active site by pyrrolidol ring of proline residue and by hydrogen bonding.

The 3-D-QSAR + CoMFA model of PEP substrates propose that the hydrophobic bonds play the essential role in substrate interaction with enzyme. This model reveals the important steric and electrostatic areas around the molecules and the presence of substituents controls the PEP activity for substrates. Analysis of obtained data allows to assume, that substrate binding in PEP active site causes essential perturbations of substrate structure. This effect mainly depends on chemical nature of the amino acid side chain, located near to proline.     

Screening of potential substrates or inhibitors of cytochrome P450 17A1 (CYP17A1) by electrochemical methods

The electrochemical redox reactions of the recombinant form of human cytochrome P450 17A1 (CYP17A1) were investigated. The hemoprotein was immobilized on the electrode modified with a biocompatible nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated using cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behavior of cytochrome P450 17A1 was performed in the presence of a natural substrate, pregnenolone (1), known inhibitor, ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetyl pregnenolone (3), cyclopregnenolone (4), and tetrabromo-pregnenolone (5). Ketoconazole, the azole inhibitor of cytochromes P450, blocked catalytic current in the presence of the substrate, pregnenolone (1). Compounds 3–5 did not demonstrate substrate properties towards the electrode/CYP17A1 system. Compound 3 did not influence catalytic activity with pregnenolone, whereas compounds 4 and 5 demonstrated some inhibitory activity. Thus, electrochemical redox reactions of CYP17A1 may serve as an adequate substitution of the reconstituted system, which requires additional redox partners for manifestation of catalytic activity of hemoproteins of the cytochrome P450 superfamily.     

Performance evaluation of a novel conceptual bioprocess for clinically-required mass production of hematopoietic cells

Objective

The novel engineered bioprocess, which was designed and modeled to provide the clinically relevant cell numbers for different therapies in our previous work (Kaleybar et al. Food Bioprod Process 122:254–268, https://doi.org/10.1016/j.fbp.2020.04.012, 2020), was evaluated by using U937 as hematopoietic model cells.

Results

The culture system showed a 30-fold expansion of U937 cells in one-step during a 10-day culture period. The cell growth profile, the substrate and oxygen consumptions, and byproduct formations were all in agreement with the model predications during 7 days. The cell proliferation decrease after 7 days was attributed to optional oxygen limiting condition in the last days of culture. The bioreactor culture system revealed also a slight enhancement of lactate dehydrogenase (LDH) production as compared to the 2D conventional culture system, indicating the low impact of shear stress on cellular damage in the dynamic system.

Conclusions

The results demonstrated that the conceptual bioprocess for suspended stem cell production has a great potential in practice although additional experiments are required to improve the system.

     

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations

BackgroundThe human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled.MethodsHere, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2 μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties.ResultsThe results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased.ConclusionsWe postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics.General significanceThe deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.     

混料设计优化黑木耳菌丝生长的农业剩余物配方

【背景】菌林矛盾日益突出,农业剩余物资源丰富,可作为食用菌栽培主要基质。【目的】筛选出适合黑木耳菌丝生长的农业剩余物配方。【方法】以大豆秸秆、油菜秸秆、玉米秸秆、花生秸秆、小麦秸秆和杂木屑等6种基质为原料,运用单纯形格子法进行配方设计,分析不同基质交互作用对黑木耳菌丝生长速率、菌丝生长指数、漆酶酶活、多酚氧化酶酶活和纤维素酶酶活的影响。【结果】在这些农业剩余物基质中,大豆秸秆基质最适合黑木耳菌丝生长,其次是油菜秸秆。3种主料共同作用可以优化出最适合黑木耳菌丝生长的基质配比。【结论】最终优化出一个适合黑木耳菌丝生长的农业剩余物配方：杂木屑49.4%、油菜秸秆16.4%、大豆秸秆12.2%、麦麸20%、蔗糖1%、CaSO₄ 1%。本研究为“以草代木”栽培黑木耳提供了理论基础。     

Novel insights into the transport mechanism of the human amino acid transporter LAT1 (SLC7A5). Probing critical residues for substrate translocation

BackgroundLAT1 (SLC7A5) is the transport competent unit of the heterodimer formed with the glycoprotein CD98 (SLC3A2). It catalyzes antiport of His and some neutral amino acids such as Ile, Leu, Val, Cys, Met, Gln and Phe thus being involved in amino acid metabolism. Interestingly, LAT1 is over-expressed in many human cancers that are characterized by increased demand of amino acids. Therefore LAT1 was recently acknowledged as a novel target for cancer therapy. However, knowledge on molecular mechanism of LAT1 transport is still scarce.MethodsCombined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes, have been adopted to unravel dark sides of human LAT1 structure/function relationships.ResultsIt has been demonstrated that residues F252, S342, C335 are crucial for substrate recognition and C407 plays a minor role. C335 and C407 cannot be targeted by SH reagents. The transporter has a preferential dimeric structure and catalyzes an antiport reaction which follows a simultaneous random mechanism.ConclusionsCritical residues of the substrate binding site of LAT1 have been probed. This site is not freely accessible by molecules other than substrate. Similarly to LeuT, K⁺ has some regulatory properties on LAT1.General significanceThe collected data represent a solid basis for deciphering molecular mechanism underlying LAT1 function.     

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

BackgoundStudying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy.MethodsWe report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (k_cat/K_m) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.ResultsAll enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.ConclusionsResults support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.General significanceResults presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.     

Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics

BackgroundM20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues.MethodsPepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods.ResultsPepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P¹ position. Peptidomimetics amastatin and actinonin occupied S¹ pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme.ConclusionsPepT preferentially hydrolyzes oligopeptides carrying alanine at P¹ position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions.General significanceThis study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.     

饲用微生物的分离鉴定及其对杏鲍菇菌糠发酵的效果

【目的】菌糠的营养素含量齐全,但纤维素含量过高是阻碍其饲料化利用的主要因素。故本研究筛选适合于发酵杏鲍菇菌糠的微生物菌株,以改善其饲用品质。【方法】首先,本研究采用纤维素-刚果红、苯胺蓝和MRS-Ca (De Man, Rogosa, Sharpe-Ca)筛选培养基,结合纤维素、木质素酶活力及抑菌活性的测定,从EM (effective microorganisms)原液发酵的杏鲍菇菌糠中分离筛选具有较强纤维素、木质素降解能力及抑菌能力的细菌/真菌。通过细菌16S rRNA和真菌18S rDNA基因序列分析确定菌株所属种属。其次,将筛选出的菌株菌液等体积混合制成复合菌剂用于固态发酵杏鲍菇菌糠。测定不同发酵时长菌糠营养成分含量以确定最佳发酵时间,并与相同工艺条件下EM原液发酵的杏鲍菇菌糠进行饲用品质比较。【结果】筛选并鉴定得到纤维素酶活性较高的特基拉芽孢杆菌(Bacillus tequilensis)菌株P11、发酵毕赤酵母(Pichia fermentans)菌株R8和马克斯克鲁维应变酵母(Kluyveromyces marxianus)菌株MU5;木质素酶活性较高的解淀粉芽孢杆菌(Bacillus amyloliquefaciens subsp.plantarum)菌株MU7;抑菌活性较高的类肠膜魏斯氏菌(Weissella paramesenteroides)菌株R4和乳酸片球菌(Pediococcus acidilactici)菌株R9。使用以上菌株复合发酵杏鲍菇菌糠7 d后,各项指标达到稳定。与EM原液发酵的杏鲍菇菌糠相比,复合菌剂发酵杏鲍菇菌糠的NDF和ADF分别显著降低了19.6%和21.44%(P0.05);CP (crude protein)、CA (crude ash)和EE (ether extract)含量分别显著提高了10.44%、5.26%和123.53%(P0.05)。【结论】本研究筛选得到的芽孢杆菌、酵母菌和乳酸菌优势菌株复合后用于发酵杏鲍菇菌糠可以很好地改善其饲用品质,效果优于生产中常用市售EM原液。     

Homology Modeling of the CG-specific DNA Methyltransferase SssI and its Complexes with DNA and AdoHcy

Abstract

Prokaryotic DNA methyltransferase M. SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed “FRankenstein's monster” approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

A scalable life cycle inventory of an automotive power electronic inverter unit—part II: manufacturing processes

Purpose

A scalable life cycle inventory (LCI) model, which provides mass composition and gate-to-gate manufacturing data for a power electronic inverter unit intended for controlling electric vehicle propulsion motors, was developed. The purpose is to fill existing data gaps for life cycle assessment (LCA) of electric vehicles. The model comprises new and easy-to-use data with sufficient level of detail to enable proper component scaling and in-depth analysis of inverter units. The aim of this article (part II) is to describe the modeling of all production steps and present new datasets. Another objective is to explain the strategies for data collection, system boundaries, and how unit process datasets were made to interact properly with the scalable design model (part I).

Methods

Data for the manufacturing of the inverter unit was collected from a variety of literature, technical specifications, factory data, site visits, and expert interviews. The model represents current levels of technology and modern industrial scale production. Industry data dates back to 2012. Some older literature is referred to, but only if it was found to remain relevant. Upstream, new data has been gathered to the point where the Ecoinvent database can be used to model a full cradle-to-gate inventory. To make the LCI model easy to use, each flow crossing the system boundary is reported with a recommended linked flow to this database.

Results and discussion

The screening and modeling of manufacturing inverter units resulted in a substantial compilation of new inventory data. In close integration with the design model, which is scalable in size over a range of 20–200 kW in nominal power and 250–700 V in DC system voltage (part I), it forms a comprehensive scalable LCI model of a typical automotive power electronic inverter unit intended for traction motor control. New production data covers electroplating of gold, electro-galvanization, machining and anodizing of aluminum, ceramic substrate fabrication, direct copper bonding, photoimaging and regenerative etching, power module assembly with a two-step soldering process, and the assembly of automotive printed circuit boards.

Conclusions

Interviews with experts were found to be vital for effective data collection and the reporting of details a key to maintaining data usability over time, for reuse, rework, and criticism by other LCA practitioners.