Day 1 :
Keynote Forum
Peter J F Henderson
Astbury Centre for Structural Molecular Biology - University of Leeds, UK
Keynote: Kinetic and molecular dissection of coupled ion-substrate membrane transport proteins
Time : 09:30-10:10
Biography:
Peter J F Henderson is a Professor of Biochemistry and Molecular Biology in the University of Leeds. He obtained his BSc in 1965 and PhD in 1968, both in Biochemistry,
at the University of Bristol. After Postdoctoral training at the Enzyme Institute, Madison, University of Wisconsin and in the Department of Biochemistry at Leicester, he
became a University Lecturer in 1973. In 1975 he moved to the Department of Biochemistry at Cambridge, where he became Reader in Molecular Biology of Membranes in
1990. He has held Visiting Professorships in Japan, Canada and Australia. He was Scientifi c Director of the European Membrane Protein (EMeP) consortium 2003-2008,
Coordinator of the European Drug Initiative for Channels and Transporters (EDICT) 2008-2012 and held Leverhulme Trust Emeritus Research Fellowships in 2001-2002
and 2014-2017. He has published over 200 scientifi c papers in the fi elds of Membrane Transport, Enzyme Kinetics and Structural Biology.
Abstract:
The Mhp1 Na+, -hydantoin membrane symport protein from Microbacterium liquefaciens is a paradigm for the nucleobasecation-
symport, NCS-1, family of transport proteins found widely in archaebacteria, bacteria, yeasts and plants. Th eir
metabolic roles include the capture by cells of nitrogen compounds and vitamins from the environment. Mhp1 is also a
structural model for the huge range of ‘5-helix-inverted-repeat’ superfamily of proteins, because, unusually, crystal structures
are available for its open-outwards, occluded, and open-inward conformations. Here we accomplish a detailed dynamic model
of the partial reactions in an alternating access cycle of membrane transport derived from substrate binding studies to the
purifi ed Mhp1 protein by combining novel mass spectrometry, stopped-fl ow and steady state kinetic analyses and mutagenesis.
Th e mechanism of coupling substrate transport to the Na+, -gradient is revealed during a sequence of mostly reversible kinetic
steps that explain how transfer of substrate across the membrane is aff ected by changes in conformational states. Th e AceI
H+/substrate antiport protein from Acinetobacter baumannii is a paradigm for the proteobacterial antimicrobial compound
effl ux (PACE) family of drug effl ux proteins found dispersed throughout the Proteobacteria. AceI contributes to the resistance
of Acinetobacter baumannii towards the widely used antiseptic, chlorhexidine. Currently there is little structural information
about the PACE family of transport proteins, but progress towards understanding the recognition of substrates and cations by
AceI and its homologues will be discussed.
Keynote Forum
Magali Remaud Simeon
INSA - Université de Toulouse, France
Keynote: Mixing enzyme discovery with engineering for sucrose-derived bioproducts: The case of GH13 and GH70 polymerases
Time : 10:10-10:50
Biography:
Magali Remaud Simeon is Professor at the National Institute of Applied Sciences of Toulouse and is head of the Catalysis and Enzyme Molecular Engineering group of
the “Laboratoire d’Ingénierie des Système Biologiques and Procédé (LISBP). She received her PhD in Biochemistry from the University of Toulouse and was Post-Doc
at the University of Pennsylvania. She has co-authored more than 150 papers and is co-inventor of 22 patents. Her research activities focus on Enzyme Engineering
for white biotechnology, green chemistry, health, food/feed industries and synthetic biology. They cover enzyme structure/activity relationship studies, kinetic resolution,
evolution combining both rational and combinatorial approaches, and applications to the synthesis of glycans, glycoconjugates and various synthons of interest. Her work
is currently focused on the search and generation of enzymes displaying new specifi cities and improved catalytic properties. Her objective is to open new trajectories for
biomass transformation. To this end, she specifi cally targets the integration of tailored enzymes in chemo-enzymatic cascades, new metabolic pathways or enzyme-based
processes.
Abstract:
The exploration of the natural diversity, through data mining, functional genomics and/or metagenomics is an effi cient mean
to discover enzymes showing new functions or improved performances. Th ese approaches can be further completed or run
in parallel with semi-rational protein engineering based on structure/function studies or directed molecular evolution inspired
from nature. Which of these alternatives are the best ones, in terms of eff ort, rapidity and effi ciency? Th is is an open question
to which a defi nite answer can be hardly formulated a priori. For illustration, we will take a few examples from our most recent
work on glucansucrases from GH13 and GH70 families. Th ese enzymes are naturally very effi cient transglucosylases. Th ey
use sucrose as substrate and catalyze polymerization of its glucosyl units as a main reaction. Depending on their specifi city,
structures varying in size as well as in glycosidic linkage types can be obtained, thus giving access to an interesting panel of
biopolymers. A campaign of genome sequencing and data mining allowed the isolation of atypical enzymes with new product
specifi cities. In particular, a hyper effi cient polymerase producing a gel-like polymer and, in contrast an enzyme synthesizing
directly from sucrose a polymer of well-controlled low molar mass could be characterized. Structure-function studies combined
with mutagenesis assays allowed us to decipher some of the molecular mechanisms behind the control of the polymer size and
enzyme processivity. Another key property of these catalysts is coming from their ability to glucosylate a broad spectrum of
hydroxylated molecules. Computational protein design, structurally-guided engineering and also random approaches such as
neutral evolution was implemented for a fi ne tuning of their acceptor specifi city toward non-natural acceptors such chemically
protected disaccharides for vaccinal applications, polyol, fl avonoids, or various chemicals. Th ese various approaches will be
described and discussed with regard to the engineering objectives.
Keynote Forum
Sergey Suchkov 2
I M Sechenov First Moscow State Medical University, Russia
Keynote: Principles of profi ling as applicable to the infrastructure of continuous education system to impact for having drug design to suit an innovative model of translational pipeline
Time : 10:00-10:40
Biography:
Sergey Suchkov graduated from Astrakhan State Medical University and awarded with MD, then in 1985 maintained his PhD at the I M Sechenov Moscow Medical
Academy and in 2001, he maintained his Doctorship Degree at the Nat Inst of Immunology, Russia. From 1987 through 1989, he was a senior Researcher, Koltzov Inst
of Developmental Biology. From 1989 through 1995, he was a Head of the Lab of Clinical Immunology, Helmholtz Eye research Institute in Moscow. From 1995 through
2004, he was a Chair of the Dept. for Clinical Immunology, Moscow Clinical Research Institute. He has been trained at: NIH; Wills Eye Hospital, PA, USA; Univ. of Florida
in Gainesville; UCSF, S-F, CA, USA; Johns Hopkins University, Baltimore, MD, USA. He was an Exe Secretary-in-Chief of the Editorial Board, Biomedical Science, an
international journal published jointly by the USSR Academy of Sciences and the Royal Society of Chemistry, UK. At present, he is a Chair, Dept. for Personal-ized and
Translational Medicine, I M Sechenov First Moscow State Medical University. He is a member of the New York Academy of Sciences, USA; American Chemical Society
(ACS), USA; American Heart Association (AHA), USA; EPMA (European Association for Predictive, Preventive and Personalized Medicine), Brussels, EU; ARVO (American
Association for Research in Vision and Ophthalmology); ISER (International Society for Eye Re-search); PMC (Personalized Medicine Coalition), Washington, USA.
Abstract:
Personalized medicine (PM) as the healthcare of the future represents an innovative model for advanced healthcare and
robust platform for relevant industrial branches for diagnostics and pharmaceutics. However, rapid market penetration
of new technologies demands the implementation of reforms not only in biopharma, but also in education. Th erefore, the
problem of the updated education of specialists in bioengineering, drug design and affi liated fi elds is becoming particularly
urgent, and it requires signifi cant revision of newer programs and curricula to be updated. Modernization and integration of
widely accepted standards require consolidation of both the natural and medical sciences that may become the conceptual
basis for the biopharma education. Th e main goal of this training is to provide development of novel multifaceted approaches
to build academic schools for future generations. So, a higher, secondary and primary education as a trio should be integrated
into the circuit. Based on current trends and own experience, we have made the fi rst steps towards reshuffl ing the canonical
educational tandem “School-University” and restructuring of specialized groups (with targeted disciplines) to get the mentees
to be involved into having the existing healthcare system advanced and stepped forward. Moreover, non-canonical approach
has been used to create a team of young researchers and biopharma students which has been recognized as Th e International
Research Team of Youngsters under the aegis of EPMA (Brussels, EU) and ISPM (Tokyo, Japan). Th e integration of the primary
and secondary education provides: 1. development in the chosen direction; and 2. optimization of the jointly set activity of a
student and the teacher within a pair or a tandem (mentor-mentee). Th e above-mentioned has pre-determining value, because
under the disintegration of the world community expressed the competition in quality of the scientifi c intellect dramatically
increases. Th e same occurs in the areas of quality of all of three segments of the educational process, i.e., pre-college (secondary
school), university and graduate.
Keynote Forum
David Rabuka
Catalent Biologics, USA
Keynote: Developing site-specifi cally modifi ed ADCs using a chemoenzymatic approach
Time : 11:50-12:30
Biography:
We have developed the SMARTagTM technology platform, which enables precise, programmable, site-selective chemical
protein modifi cation. Leveraging the target sequence of formylglycine generating enzyme (FGE), we chemoenzymatically
modify proteins to generate a precisely placed aldehyde functionality that can be chemically elaborated. Subsequently, novel
ligation chemistry is employed that exploits this “aldehyde tag” site. We will present recent data on our novel protein modifi cation
platform and its application to generating novel bioconjugates, including ADCs, utilizing our new conjugation chemistries and
linkers. Th e application of these chemistries to generate site-specifi cally modifi ed bioconjugates with improved effi cacy and
safety profi les will be presented. Additionally, we will highlight the progress in developing conjugates with a focus on preclinical
studies as well as highlight our progress in cell line development and manufacturing by using this chemoenzymatic approach.
Abstract:
David Rabuka received a PhD in Chemistry at the University of California, Berkeley as a Chevron Fellow in the Lab of Carolyn Bertozzi. His research included developing
and applying the SMARTagTM platform technology to cell surface modifi cation. Prior to joining Bertozzi’s lab, he worked at the Burnham Institute synthesizing complex
glycans followed by Optimer Pharmaceuticals, where he focused on the development of glycan and macrolide based antibiotics. He was CSO, President and Co-founder
of Redwood Bioscience, where he developed novel protein conjugation methods and biotherapeutic applications such as antibody-drug conjugates. Redwood Bioscience
was acquired by Catalent Pharma Solutions in Oct 2014, where he has continued to apply the SMARTagTM technology with various collaborators and partners as a Global
Head of R&D. He graduated with a Double Honors BS in Chemistry and Biochemistry from the University of Saskatchewan, where he received the Dean’s Science Award,
and holds an MS in Chemistry From the University of Alberta. He has authored over 45 major publications, as well as numerous book chapters and holds over 30 patents.
- Industrial Biotechnology| Pharmaceutical Biotechnology |Biotechnology Applications
Location: Armstrong
Chair
Sergey Suchkov
I M Moscow State Medical University, Russia
Session Introduction
Iwona Zur
Polish Academy of Sciences, Poland
Title: Efficiency of the antioxidative system is the first prerequisite for effective doubled haploids production with the use of isolated microspore culture method
Biography:
Iwona Å»ur completed her PhD and Habilitation in the fi eld of Agronomy and Plant Physiology at the University of Agriculture in Kraków, Poland. Since 2010, she has
been the Head of the Department of Cell Biology at the Institute of Plant Physiology Polish Academy of Sciences. She has published 38 papers in peer-reviewed
journals.
Abstract:
The technology of doubled haploids as the fastest route to total homozygosity is highly appreciated in many domains of basic research
and breeding. Among several methods, the one using isolated and in vitro-cultured immature cells of male gametophyte induced
towards embryogenic development (microspore embryogenesis-ME) possesses the highest potential for commercial application.
However, effi cient ME induction requires a precisely balanced stress treatment, strong enough to induce microspore reprogramming
but not exceeding cell stress tolerance threshold. As the general cause of injuries in in vitro-cultured cells is the overproduction of
reactive oxygen species (ROS), an effi cient antioxidative defence was suggested as the fi rst prerequisite for stress survival and eff ective
ME initiation. To establish the role of ROS and the antioxidative system in ME initiation, the generation of hydrogen peroxide, and
the activities of antioxidative enzymes and low molecular weight antioxidants were analysed in isolated microspores of two cultivars
of barley (Hordeum vulgare L.), winter cv. Igri and spring cv. Golden Promise, diff ering signifi cantly with respect to embryogenic
potential. Th e analyses were conducted in microspores redirected towards embryogenic development by low temperature tillers pretreatment
(4 weeks at 4°C). Additionally, the eff ects of compounds known as cellular redox status modifi ers, e.g. glutathione and L-2-
oxo-4-thiazolidinecarboxylic acid (OTC), on microspore viability and ME initiation effi ciency were estimated. Th e received results
suggest that the activity of the antioxidative system is the fi rst prerequisite for successful ME initiation, though in the case of its low
activity, antioxidative defence could be supported by the application of exogenous antioxidants.
Omirbekova N Zh
Al-Farabi Kazakh National University, Kazakhstan
Title: Development of approach to obtain Brachypodium distachyon L. regenerative plants with morphogenetic stability
Biography:
Omirbekova N Zh graduated from Al-Farabi Kazakh National University and Lomonosov Moscow State University and has completed her Doctoral studies from
Al-Farabi Kazakh National University. She is currently a Professor at the Department of Molecular Biology and Genetics, School of Biology and Biotechnology
of KazNU named after Al-Farabi (Republic of Kazakhstan). Her research interests include chemical mutagenesis, genetics and biochemistry of wheat. She has
published more than 30 papers in high valued journals.
Abstract:
The aim of the research is development of eff ective methodological approaches of in vitro cultivation, object 21 line (BD21) B.
distachyon. In order to develop cultivation methods, ability for callus formation, regeneration of generative and vegetative organs
of VD21 was studied. To cultivate, Linsmayer-Skoog and Murashige-Skoog medium, additional introduction of phytohormones
was used. Aseptic culture conditions for callusogenesis cultivation: under dark conditions at a temperature of 24°C, for t shoots
regeneration: 16/8 hour photoperiod and lighting of 3000 lux. Infl orescence and immature embryos isolated from green spikes of
vegetating plants and isolated embryos from mature seeds were used as primary explants to induce callus formation in vitro. During
immature embryo cultivation, callus formation takes place near the corimbe for 20-25 days. During the cultivation of whole caryopsis
with mature embryos, the sprouts grew aft er a week of cultivation on MS medium without hormones. Th e level of maturity of isolated
caryopsis has a signifi cant infl uence on the callus formation and the type of callus tissue. Th e mature caryopsis formed callus on
the 10th day of cultivation with a frequency of 75%. Th e cultivation of the overgrown caryopsis in the dark on medium MS 1 with
2 mg/L 2.4 DPA, led to the formation of a primary shoot in 60% of explants; the formation of callus in the area of the scute, but for
30-35 days. Passage of the callus on the same medium and on the hormone-free medium led to the appearance of greenish pointwise
impregnation of 30% of the calluses. For microclonal propagation, nodal segments of young shoots of plants were introduced into the
culture. To culture introduction, side shoots 5 cm long with 3-4 interstitial sites were cut, the microcrops were planted in inducing
media. Th e shoot-forming capacity of primary explants was about 59%; the multiplication factor for two passages was 5.7.
Biography:
Helmi Mohamed El-bendary is Assistant Professor of Agriculture at Fayoum University. He fi nished his BSc in Plant Protection Department at Cairo University and D.S.P.U at Mediterranean Agronomic Institute, Greece, M.S.c. at Cairo University, LLB at Cairo University, and Ph.D. at Mansoura University.
Abstract:
Naturally occurring micron sized silica has gained enormous popularity as a physically active insecticide. Nano-sized silica has insecticidal properties and would be needed in lesser quantity in comparison with conventional insecticides because of the huge surface to volume ratio of nanoparticles. Nano molecules have been widely used in consumer and industrial applications,
such as medicine, cosmetics and foods, because they exhibit unique physicochemical properties and innovative functions. However,
nanomaterials (NMs) can also be problematic in terms of eliciting a toxicological eff ect by their small size. Th e present study was
designed to examine the toxic eff ects of orally administered pesticide Sil-MATRIX 29% (potassium silicate) and silica nano-particles
(SiO2-NPs) using male albino rats, at sublethal doses [2/5, 1/4 and 1/8 LD50], relative to control on [body, organs weight such as liver, kidney, heart, spleen, and cytotoxic eff ect (such as total protein content levels as biochemical aspects)] for 28 and 45 days’ time exposure period. Orally ingested Sil-MATRIX 29% and silica nanoparticles (SiO2-NPs) [2/5, 1/4 and 1/8 LD50] were not associated with signifi cant changes in the average gain of body and organ weight. On the other hand, total protein content value aft er ingestion
with Sil-MATRIX and SiO2-NPs for all doses and treatments time period were increased signifi cantly in a pattern similar to control rats. Our results suggested that the well-dispersed nano-silica cytotoxic eff ect caused systemic exposure in mouse and induced mutagenic activity. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and safer form of (NMs).
Jean M Francois
Université Fédérale Toulouse Midi-Pyrénées, France
Title: Engineering carbon-conserving synthetic pathways for assimilation and conversion of C5/C6 carbon sources into added value chemicals
Biography:
Jean M François got his PhD in Biological Science and Agronomy from the University of Louvain (Belgium) in 1988. He is Professor of Industrial Microbiology and
BioNanotechnology at the Federal University of Toulouse, School of Engineer. His research activity concerns integrated physiology and functional genomics in
microbial systems, with a specifi c focus on carbon and energy metabolism in yeast and fi lamentous fungi . He is author of more than 180 papers and 15 patents
and Editor in Chief of BMC Biotechnology for Biofuels.
Abstract:
The development of carbon effi cient pathways for added value (bio)chemicals production is the essence of White Biotechnology.
Th e limit of carbon conservation in all (bio)chemical syntheses is determined by the electron balance in substrate(s) and
product(s). Frequently, natural metabolic networks do not have the stoichiometric capacity to produce a value-added compound
at yields that correspond to the thermodynamic maximum. A good example of natural metabolic networks lacking stoichiometric
effi ciency is the bioproduction of glycolic acid (GA), a two carbon compound of considerable industrial interest notably in cosmetics
and biodegradable polymers. We addressed this objective to approach this maximal conversion yield by employing the following
strategies. Firstly, we reconsider a completely diff erent route of C5 assimilation that by-passes the decarboxylation reaction in the
pentose phosphate pathway and that rely on the carbon-conserving aldolytic cleavage of X1P or R1P to yield the C2 compound
glycolaldehyde and the C3 DHAP compound. Th is metabolic scheme required the expression of human hexo(fructo)kinase(Khk-C)
and human aldolase (Aldo-B). Th en glycoaldehyde can be either reduced by endogenous aldehyde reductase to produce ethylene
(EG) glycol or oxidized into glycolic acid. With this approach, we obtained yield of EG and GA close to maximal theoretical yield of 1
mol/ mol sugar. Interestingly, we found that the engineered strain expressing this synthetic pathway exhibited a remarkable rewiring
of the metabolic networks that culminate with a dramatic reduced metabolites and metabolic energy levels. We then combined this
synthetic pathway with the natural glyoxylate shunt that can be engineered to produce GA from DHAP. Th is combination led to
an optimized production strain that produced ~30 % more GA from a xylose/glucose mixture (66%/33%) than when the natural
pathway is working alone.
Jean M Francois
Université Fédérale Toulouse Midi-Pyrénées, France
Title: Engineering carbon-conserving synthetic pathways for assimilation and conversion of C5/C6 carbon sources into added value chemicals
Biography:
Jean M François got his PhD in Biological Science and Agronomy from the University of Louvain (Belgium) in 1988. He is Professor of Industrial Microbiology and
BioNanotechnology at the Federal University of Toulouse, School of Engineer. His research activity concerns integrated physiology and functional genomics in
microbial systems, with a specifi c focus on carbon and energy metabolism in yeast and fi lamentous fungi . He is author of more than 180 papers and 15 patents
and Editor in Chief of BMC Biotechnology for Biofuels.
Abstract:
The development of carbon effi cient pathways for added value (bio)chemicals production is the essence of White Biotechnology.
Th e limit of carbon conservation in all (bio)chemical syntheses is determined by the electron balance in substrate(s) and
product(s). Frequently, natural metabolic networks do not have the stoichiometric capacity to produce a value-added compound
at yields that correspond to the thermodynamic maximum. A good example of natural metabolic networks lacking stoichiometric
effi ciency is the bioproduction of glycolic acid (GA), a two carbon compound of considerable industrial interest notably in cosmetics
and biodegradable polymers. We addressed this objective to approach this maximal conversion yield by employing the following
strategies. Firstly, we reconsider a completely diff erent route of C5 assimilation that by-passes the decarboxylation reaction in the
pentose phosphate pathway and that rely on the carbon-conserving aldolytic cleavage of X1P or R1P to yield the C2 compound
glycolaldehyde and the C3 DHAP compound. Th is metabolic scheme required the expression of human hexo(fructo)kinase(Khk-C)
and human aldolase (Aldo-B). Th en glycoaldehyde can be either reduced by endogenous aldehyde reductase to produce ethylene
(EG) glycol or oxidized into glycolic acid. With this approach, we obtained yield of EG and GA close to maximal theoretical yield of 1
mol/ mol sugar. Interestingly, we found that the engineered strain expressing this synthetic pathway exhibited a remarkable rewiring
of the metabolic networks that culminate with a dramatic reduced metabolites and metabolic energy levels. We then combined this
synthetic pathway with the natural glyoxylate shunt that can be engineered to produce GA from DHAP. Th is combination led to
an optimized production strain that produced ~30 % more GA from a xylose/glucose mixture (66%/33%) than when the natural
pathway is working alone.
- Structural Enzymology | Enzymology & Biochemistry
Location: Armstrong
Chair
Magali Remaud-Simeon
INSA-Tolouse, France
Session Introduction
Albert Jeltsch
University Stuttgart, Germany
Title: Molecular enzymology of DNA methyltransferases – conformational changes and allosteric regulation
Biography:
Albert Jeltsch completed his PhD working on the mechanism of restriction endonucleases at University of Hannover in 1994. Afterwards, he started to study
DNA methyltransferases at Justus-Liebig University Giessen and at Jacobs University Bremen. Since 2011, he is a Professor of Biochemistry at the University
Stuttgart. He received the Gerhard-Hess award (DFG) and BioFuture award (BMBF). He has long standing expertise in Biochemical study of DNA and protein
methyltransferases, methyl lysine reading domains and in rational and evolutionary protein design. His work has been published in more than 250 publications in
peer reviewed journals and he is in the editorial boards of several journals.
Abstract:
DNA methylation is an essential epigenetic chromatin modifi cation. Th e setup and maintenance of DNA methylation patterns
depends on the coordinated activity of DNA methyltransferases (DNMTs) and their allosteric regulation by interacting proteins,
other chromatin modifi cations and post-translational modifi cations. I will present novel assays for DNMTs including single enzyme
assays to study their mechanism and conformationally locked mutants to study allosteric eff ects. Based on this, recent data regarding
the regulation and targeting of DNMTs by allosteric eff ect will be presented. Moreover, I will present insights into the mechanism of
DNMTs regarding target site location, specifi city and processivity.
David J Merkler
University of South Florida, USA
Title: N-acyltransferases and their role in fatty acid amide biosynthesis
Biography:
David J Merkler obtained a PhD in Biochemistry from Pennsylvania State University in 1985 and completed Postdoctoral Fellowships in Enzymology at Temple
University School of Medicine (1985-1987) and the Albert Einstein College of Medicine (1987-1989). His next position was as Senior Scientist at Unigene
Laboratories, Inc. involved in the in vitro production of a peptide hormone, calcitonin. In 1995, he moved back to academia as a Professor of Chemistry and
Biochemistry fi rst at Duquesne University (1995-1999) and then the University of South Florida (1999-present). His laboratory has been interested in the fatty
amides: identifi cation and characterization of the fatty acid amides (Lipidomics), identifi cation and characterization of the enzymes of fatty acid amide biosynthesis
(Enzymology and Structural Biology), and changes in the fatty acid amidome after targeted enzyme knock-out (subtraction lipidomics).
Abstract:
Fatty acid amides are a family of cell signaling lipids with the general structure of R-CO-NH-Y. Th is structural simplicity belies
a wealth of diversity amongst this lipid family as the R-group is derived from fatty acids (R-COOH) and the Y-group is derived
from biogenic amines (H2N-Y). Th e fatty acid amide family is divided into classes, defi ned by parent amines. Examples include the
N-acylethanolamines (NAEs, R-CO-NH-CH2-CH2OH) and the N-acylglycines (NAGs, R-CO-NH-CH2-COOH). Other classes of
fatty acid amides are known. Th e best known fatty acid amide is N-arachidonoylethanolamine (anandamide), a fatty acid amide found
in the human brain that binds to the cannabinoid receptors. We have a long interest in the enzymes of fatty acid amide biosynthesis.
We identifi ed an enzyme that oxidizes the NAGs to the primary fatty acid amides and showed that inhibiting this enzyme led to the
cellular accumulation of the NAGs. We have characterized several insect N-acyltransferases (from D. melanogaster, B. mori, and T.
castaneum) that catalyze the acyl-CoA-dependent formation of fatty acid amides from an amine acyl-acceptor substrate. Knock-out
experiments in D. melanogaster validate our in vitro substrate specifi c studies demonstrating that one novel N-acyltransferases, arylalkyl
N-acyltransferase-like 2 (AANATL2), does catalyze the formation of N-acyldopamines in vivo. We developed a straightforward
platform technology to rapidly identify substrates for our panel of uncharacterized insect N-acyltransferases. Our application of this
technology leads to identifi cation of an enzyme in D. melanogaster, agmatine N-acetyltransferase (AgmNAT), which catalyzes the
formation of N-acetylagmatine, a virtually unknown metabolite. We have determined the X-ray structure of AgmNAT. Our work on
AgmNAT hints at an unknown reaction in arginine metabolism and points to a novel class on fatty acid amides, the N-acylagmatine.
Th e presentation will also include our results on the kinetic and chemical mechanisms of the novel N-acyltransferases.
Karlo M Lopez
California State University-Bakersfield, USA
Title: Lysyl oxidase: A versatile and elusive enzyme
Biography:
Karlo M Lopez is currently an Associate Professor of Biochemistry at California State University, Bakersfi eld. He received a PhD from Clark University and was a
Howard Medical Institute Fellow at Pomona College. His research focuses primarily on the structural characterization of lysyl oxidase and understanding the role
this enzyme plays in cancer metastasis. He is a member of the Committee on Ethics of the American Chemical Society and was part of the Task Force for Safety
Education Guidelines.
Abstract:
Lysyl oxidase is an extracellular matrix, copper-dependent, amine oxidase that catalyzes a key crosslinking step in collagen and
elastin. Th e enzyme is synthesized as a proenzyme that, upon excretion to the extracellular matrix, is cleaved at the Gly168-
Asp169 bond by procollagen C-proteinase in the mammalian form of the enzyme. Lysyl oxidase is highly regulated and changes in its
regulation have been shown to play a role in fi brosis and several other diseases. More recently, the enzyme has been shown to play a
paradoxical role in cancer. In the early stages of cancer, the cleaved pro-peptide has been shown to inhibit the RAS oncogene, whereas
in late stages of cancer lysyl oxidase has been shown to promote metastasis. Lysyl oxidase is highly insoluble and this has hampered
its full characterization. Recent work in the by our study group has addressed some of the issues associated with the insolubility and
characterization of the enzyme. In particular, this talk will address how plasmids were used to increase enzyme yields over those
obtained directly from bovine aortic tissue, the role solubility tags play on enzyme activity and suitability for characterization studies,
and will end with an innovative new approach to drug delivery that targets lysyl oxidase in cancer cells but remains inactive in normal
cells.
Biography:
Anna V Hine studied at the University of Manchester (UK) and Harvard Medical School. She is a Reader and Associate Dean Enterprise at Aston University (UK).
In March 2013, she was named BBSRC Commercial Innovator of the Year 2013, for her work in transferring ProxiMAX randomization into SME Isogenica Ltd. She
is a Molecular Biologist by training, she relishes interdisciplinary work.
Abstract:
ProxiMAX randomization is the technology that lies behind Isogenica’s Colibra™ off ering. It is a defi ned saturation mutagenesis
process that delivers precision control of both identity and relative ratio of amino acids at specifi ed locations within a protein/
antibody library. Th us unwanted amino acids such as cysteine and methionine can be eliminated from libraries because no constraints
are imposed by the genetic code. Moreover, the process is non-degenerate, which means that encoding DNA libraries are as small as is
physically possible. ProxiMAX relies on a process of saturation cycling comprising ligation, amplifi cation and digestion for each cycle
and is the science behind the commercial Colibra™ technology. Currently focused on antibody libraries but with achieved diversities
of >99% (6 & 11 saturated codons) and the potential to generate libraries of up to 1014 components, we contest that ProxiMAX
randomization is a vital tool in engineering any protein library of the highest quality. Th is presentation will examine the development
of the ProxiMAX process and give examples of libraries created to date.
Sotaro Fujii
Hiroshima University, Japan
Title: Stability and function of a thermophilic cytochrome c’
Biography:
Sotaro Fujii is working on the stability, structure, and function of proteins that are important for microbial energy metabolism. A characteristic aspect of his research
activity is comparison of the homologous proteins isolated from microorganisms living in extreme environments in which humans cannot live and those isolated
from ‘normal’ environments.
Abstract:
Cytochromes c' are classifi ed as heme proteins found in restricted Gram-negative bacteria. Th ey usually form a homo dimeric
structure, and the single subunit typically consists of four helix bundle. Biochemical analysis showed that they can bind diatomic
gasses such as NO or CO, but not O2. Recently we purifi ed cytochrome c' from thermophilic Hydrogenophilus thermoluteolus, and
named it PHCP. H. thermoluteolus grows optimally at 52°C, indicating that PHCP is more stable than homologous proteins from
mesophiles. In this study, we compared stability and function of PHCP with its mesophilic homologue, Allochromatium vinosum
cytochrome c' (AVCP) having 55 % amino acid sequence identity. In order to check the stability, we measured the circular dichroism
spectra with increasing temperature. Th e denaturation temperature of PHCP was 87°C, which was higher than that of AVCP
(52°C). Th e X-ray structure comparison between PHCP and AVCP revealed that the stability diff erence was due to the heme-related
interactions and subunit-subunit interactions, which was also proofed by mutagenesis study. Th ese results indicated that PHCP
advantageously retains the native structure at high temperature. Th e PHCP X-ray structure further revealed a ligand binding channel
and a penta-coordinated heme, as observed in the AVCP protein, indicating PHCP could bind diatomic gasses at high temperature.
Th us, we measured the gas binding affi nity of PHCP and AVCP using absorption spectra. Th e association constant (Ka) of PHCP
with CO was 3 times lower than that of AVCP at 25°C, and PHCP could maintain normal spectral changes up to 60°C. In AVCP, such
spectral changes with CO could not to be detected at 60°C, because of denaturation of AVCP. In conclusion, PHCP has a structure
fulfi lling the requirement for both gas-binding function and thermal stability. Th is stable cytochrome c' will become a model for
protein engineering fi eld.