Repurposing Biology through Synthetic Enzymology – For Human and Planetary Health

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Cannabinoid biosynthesis using non-canonical enzymes

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Controlling enantioselectivity of limonene synthases by exploring natural diversity combined to molecular engineering

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Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family’s rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions

Building a global alliance of biofoundries (vol 10, 2040, 2019)

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment

Evolution of Enzymatic Activities in the OMPDC Suprafamily: Discovery, Design and Evolution of 3-Keto-L-Gulonate 6-Phosphate Decarboxylase

225 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) was discovered to be involved in the fermentative utilization of L-ascorbate by enteric bacteria such as E. coli K-12. KGPDC is a new member of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, and its discovery expands the range of reactions catalyzed by members of the suprafamily beyond non-cofactor-assisted decarboxylation (avoidance of a vinyl anion intermediate in the context of OMPDC), aldol-condensation (through divalent metal-ion stabilization of an enediolate intermediate in the context of HPS), and metal-independent epimerization (through active site stabilization of an enediolate intermediate in the context of RPE). The mechanism of the KGPDC-catalyzed reaction was determined, and it involves Mg2+-stabilization of an enediolate intermediate and a unique proton-relay system that provides the proton source for catalysis. KGPDC, like members of the OMPDC suprafamily, are dimers of (beta/alpha)8-barrels; the active site is located at the C-terminal ends of the beta-strands and comprises of residues originating from both monomers. The positions of functional groups are conserved amongst suprafamily members, although active site residues differ both in identity and mechanistic utility: in OMPDC, a conserved Lys at the ends of the 2nd beta-strand is involved in a charge-relay network that is required for catalysis; in KGPDC, a conserved Glu at the same position is recruited to stabilize a Mg 2+-ion that is required for stabilization of the ensuing enediolate intermediate during catalysis. By rational design, the promiscuity of the KGPDC scaffold towards catalysis of the HPS reaction was enhanced: three substitutions of E112D, R139V and T169A resulted in a 260-fold increase in catalytic efficiency, while the physiological KGPDC activity was decreased by 30-fold. These studies provided an initial insight into the opportunistic ways of Nature in bridging between points along the evolutionary landscape through the use of an active site architecture that is capable of catalyzing a reaction that has neither a semblance of chemical mechanism nor binding specificity to that of the progenitorial reaction.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Utilization of l-Ascorbate by Escherichia coli K-12: Assignments of Functions to Products of the yjf-sga and yia-sgb Operons

Escherichia coli K-12 can ferment l-ascorbate. The operon encoding catabolic enzymes in the utilization of l-ascorbate (ula) has been identified; this operon of previously unknown function had been designated the yif-sga operon. Three enzymes in the pathway that produce d-xylulose 5-phosphate have been functionally characterized: 3-keto-l-gulonate 6-phosphate decarboxylase (UlaD), l-xylulose 5-phosphate 3-epimerase (UlaE), and l-ribulose 5-phosphate 4-epimerase (UlaF). Several products of the yia-sgb operon were also functionally characterized, although the substrate and physiological function of the operon remain unknown: 2,3-diketo-l-gulonate reductase (YiaK), 3-keto-l-gulonate kinase (LyxK), 3-keto-l-gulonate 6-phosphate decarboxylase (SgbH), and l-ribulose 5-phosphate 4-epimerase (SgbE)

www.mdpi.com/journal/ijms Development of Quorum-Based Anti-Virulence Therapeutics Targeting Gram-Negative Bacterial Pathogens

Abstract: Quorum sensing is a cell density-dependent signaling phenomenon used by bacteria for coordination of population-wide phenotypes, such as expression of virulence genes, antibiotic resistance and biofilm formation. Lately, disruption of bacterial communication has emerged as an anti-virulence strategy with enormous therapeutic potential given the increasing incidences of drug resistance in pathogenic bacteria. The quorum quenching therapeutic approach promises a lower risk of resistance development, since interference with virulence generally does not affect the growth and fitness of the bacteria and, hence, does not exert an associated selection pressure for drug-resistant strains. With better understanding of bacterial communication networks and mechanisms, many quorum quenching methods have been developed against various clinically significant bacterial pathogens. In particular, Gram-negative bacteria are an important group of pathogens, because, collectively, they are responsible for the majority of hospital-acquired infections. Here, we discuss the current understanding of existing quorum sensing mechanisms and present important inhibitory strategies that have been developed against this group of pathogenic bacteria

Biologically engineered microbes for bioremediation of electronic waste: Wayposts, challenges and future directions

10.1049/enb2.12020Engineering Biology6123-3