Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the substrate P2' position. Beside illustrating the role played by P1 and P2' residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities.

Multiscale simulations identify origins of differential carbapenem hydrolysis by the OXA-48 β-lactamase

OXA-48 β-lactamases are frequently encountered in bacterial infections caused by carbapenem-resistant Gram-negative bacteria. Due to the importance of carbapenems in treatment of healthcare-associated infections, and the increasingly wide dissemination of OXA-48-like enzymes on plasmids, these β-lactamases are of high clinical significance. Notably, OXA-48 hydrolyses imipenem more efficiently than other commonly used carbapenems, such as meropenem. Here, we use extensive multi-scale simulations of imipenem and meropenem hydrolysis by OXA-48 to dissect the dynamics and to explore differences in reactivity of the possible conformational substates of the respective acylenzymes. QM/MM simulations of the deacylation reaction for both substrates demonstrate that deacylation is favoured when the 6α-hydroxyethyl group is able to hydrogen bond to the water molecule responsible for deacylation, but disfavoured by increasing hydration of either oxygen of the carboxylated Lys73 general base. Differences in free energy barriers calculated from the QM/MM simulations correlate well with the experimentally observed differences in hydrolytic efficiency between meropenem and imipenem. We conclude that the impaired breakdown of meropenem, compared to imipenem, which arises from a subtle change in the hydrogen bonding pattern between the deacylating water molecule and the antibiotic, is most likely induced by the meropenem 1-methyl group. In addition to increased insights into carbapenem breakdown by OXA β-lactamases, which may aid design of new antibiotics or inhibitors, our approach exemplifies the combined use of atomistic simulations in determining the possible different enzyme-substrate substates, and their influence on enzyme reaction kinetics.

Teachers’ Misunderstandings that Affect the Learning of Their Pasifika Students

<p><b>Much current Pasifika research has a focus on looking at traditional Pasifika ways of learning to find an answer for low achievement among Pasifika students. Non-Pasifika research seems to see the solution as entailing teachers learning about their Pasifika students' lives. Yet neither of these approaches seems to make a difference for Pasifka student achievement. This study has shown that what does make a difference is the employment of good pedagogy by teachers who like and believe in their Pasifka students' abilities to succeed in the palagi education system. This study has also shown that what impedes progress for Pasifika students' achievement is the beliefs that teachers hold about "Pasifika ways of learning".</b></p> <p>For two days per week over a six week period a group of Year 9 Pasifika students were observed across a range of their classes in a medium sized, urban, low decile college which has a high proportion of Pasifika students. Observations and focus group interviews with Pasifika students and their teachers were conducted to explore the Pasifika student's engagement level and learning. A research methodology of mediated dialogue allowed the participants to be heard as authorities on their own experiences. The Pasifika students and their teachers were supported to hear the meanings each had given to the words and actions observed in the classroom. The Pasifika students were involved in the research as they co-constructed [with the researcher as scribe] the information they wanted their teacher to know. Teachers were able to respond to their Pasifika students' words and the Pasifika students were able to hear their teachers' responses.</p> <p>The findings were shaped as four vignettes and interpreted using the metaphor of an enzyme reaction. Each vignette described the type of learning and different engagement levels observed in a specific classroom. The vignettes included three classrooms where: students were not engaged at all with their learning because the relationship between the teacher and the students was poor; there was an appropriate relationship between the teacher and the students but the teachers' practice was poor; and the relationship between the students and the teacher was good but the teacher's expectations of the Pasifika students were low, and Pasifika learning was poor. In one vignette the relationship between the teacher and the students was one of trust and the teacher used teaching strategies that engaged and challenged the Pasifika students. In this classroom Pasifika learning was happening for a time.</p> <p>The study found that good Pasifika learning requires that the teacher must have all three of the following teaching strategies: allowing Pasifika students respect as a learner; being able to scaffold Pasifika learning at the right level and engaging their Pasifika students in active learning. The Pasifika student must have confidence and trust in their teacher to engage with the teacher in the active pursuit of learning. The classroom teacher must also have confidence in the Pasifika student's ability e.g., high expectations. If any of the parts described above are missing Pasifika learning is poor. The study demonstrates that the ways in which teachers fall into poor relationships and poor teaching practices can be related to their beliefs about Pasifika values and "Pasifika ways of learning".</p>

Teachers’ Misunderstandings that Affect the Learning of Their Pasifika Students

<p><b>Much current Pasifika research has a focus on looking at traditional Pasifika ways of learning to find an answer for low achievement among Pasifika students. Non-Pasifika research seems to see the solution as entailing teachers learning about their Pasifika students' lives. Yet neither of these approaches seems to make a difference for Pasifka student achievement. This study has shown that what does make a difference is the employment of good pedagogy by teachers who like and believe in their Pasifka students' abilities to succeed in the palagi education system. This study has also shown that what impedes progress for Pasifika students' achievement is the beliefs that teachers hold about "Pasifika ways of learning".</b></p> <p>For two days per week over a six week period a group of Year 9 Pasifika students were observed across a range of their classes in a medium sized, urban, low decile college which has a high proportion of Pasifika students. Observations and focus group interviews with Pasifika students and their teachers were conducted to explore the Pasifika student's engagement level and learning. A research methodology of mediated dialogue allowed the participants to be heard as authorities on their own experiences. The Pasifika students and their teachers were supported to hear the meanings each had given to the words and actions observed in the classroom. The Pasifika students were involved in the research as they co-constructed [with the researcher as scribe] the information they wanted their teacher to know. Teachers were able to respond to their Pasifika students' words and the Pasifika students were able to hear their teachers' responses.</p> <p>The findings were shaped as four vignettes and interpreted using the metaphor of an enzyme reaction. Each vignette described the type of learning and different engagement levels observed in a specific classroom. The vignettes included three classrooms where: students were not engaged at all with their learning because the relationship between the teacher and the students was poor; there was an appropriate relationship between the teacher and the students but the teachers' practice was poor; and the relationship between the students and the teacher was good but the teacher's expectations of the Pasifika students were low, and Pasifika learning was poor. In one vignette the relationship between the teacher and the students was one of trust and the teacher used teaching strategies that engaged and challenged the Pasifika students. In this classroom Pasifika learning was happening for a time.</p> <p>The study found that good Pasifika learning requires that the teacher must have all three of the following teaching strategies: allowing Pasifika students respect as a learner; being able to scaffold Pasifika learning at the right level and engaging their Pasifika students in active learning. The Pasifika student must have confidence and trust in their teacher to engage with the teacher in the active pursuit of learning. The classroom teacher must also have confidence in the Pasifika student's ability e.g., high expectations. If any of the parts described above are missing Pasifika learning is poor. The study demonstrates that the ways in which teachers fall into poor relationships and poor teaching practices can be related to their beliefs about Pasifika values and "Pasifika ways of learning".</p>

Teachers' Misunderstandings that Affect the Learning of Their Pasifika Students

<p>Much current Pasifika research has a focus on looking at traditional Pasifika ways of learning to find an answer for low achievement among Pasifika students. Non-Pasifika research seems to see the solution as entailing teachers learning about their Pasifika students' lives. Yet neither of these approaches seems to make a difference for Pasifka student achievement. This study has shown that what does make a difference is the employment of good pedagogy by teachers who like and believe in their Pasifka students' abilities to succeed in the palagi education system. This study has also shown that what impedes progress for Pasifika students' achievement is the beliefs that teachers hold about "Pasifika ways of learning". For two days per week over a six week period a group of Year 9 Pasifika students were observed across a range of their classes in a medium sized, urban, low decile college which has a high proportion of Pasifika students. Observations and focus group interviews with Pasifika students and their teachers were conducted to explore the Pasifika student's engagement level and learning. A research methodology of mediated dialogue allowed the participants to be heard as authorities on their own experiences. The Pasifika students and their teachers were supported to hear the meanings each had given to the words and actions observed in the classroom. The Pasifika students were involved in the research as they co-constructed [with the researcher as scribe] the information they wanted their teacher to know. Teachers were able to respond to their Pasifika students' words and the Pasifika students were able to hear their teachers' responses. The findings were shaped as four vignettes and interpreted using the metaphor of an enzyme reaction. Each vignette described the type of learning and different engagement levels observed in a specific classroom. The vignettes included three classrooms where: students were not engaged at all with their learning because the relationship between the teacher and the students was poor; there was an appropriate relationship between the teacher and the students but the teachers' practice was poor; and the relationship between the students and the teacher was good but the teacher's expectations of the Pasifika students were low, and Pasifika learning was poor. In one vignette the relationship between the teacher and the students was one of trust and the teacher used teaching strategies that engaged and challenged the Pasifika students. In this classroom Pasifika learning was happening for a time. The study found that good Pasifika learning requires that the teacher must have all three of the following teaching strategies: allowing Pasifika students respect as a learner; being able to scaffold Pasifika learning at the right level and engaging their Pasifika students in active learning. The Pasifika student must have confidence and trust in their teacher to engage with the teacher in the active pursuit of learning. The classroom teacher must also have confidence in the Pasifika student's ability e.g., high expectations. If any of the parts described above are missing Pasifika learning is poor. The study demonstrates that the ways in which teachers fall into poor relationships and poor teaching practices can be related to their beliefs about Pasifika values and "Pasifika ways of learning".</p>

Teachers' Misunderstandings that Affect the Learning of Their Pasifika Students

<p>Much current Pasifika research has a focus on looking at traditional Pasifika ways of learning to find an answer for low achievement among Pasifika students. Non-Pasifika research seems to see the solution as entailing teachers learning about their Pasifika students' lives. Yet neither of these approaches seems to make a difference for Pasifka student achievement. This study has shown that what does make a difference is the employment of good pedagogy by teachers who like and believe in their Pasifka students' abilities to succeed in the palagi education system. This study has also shown that what impedes progress for Pasifika students' achievement is the beliefs that teachers hold about "Pasifika ways of learning". For two days per week over a six week period a group of Year 9 Pasifika students were observed across a range of their classes in a medium sized, urban, low decile college which has a high proportion of Pasifika students. Observations and focus group interviews with Pasifika students and their teachers were conducted to explore the Pasifika student's engagement level and learning. A research methodology of mediated dialogue allowed the participants to be heard as authorities on their own experiences. The Pasifika students and their teachers were supported to hear the meanings each had given to the words and actions observed in the classroom. The Pasifika students were involved in the research as they co-constructed [with the researcher as scribe] the information they wanted their teacher to know. Teachers were able to respond to their Pasifika students' words and the Pasifika students were able to hear their teachers' responses. The findings were shaped as four vignettes and interpreted using the metaphor of an enzyme reaction. Each vignette described the type of learning and different engagement levels observed in a specific classroom. The vignettes included three classrooms where: students were not engaged at all with their learning because the relationship between the teacher and the students was poor; there was an appropriate relationship between the teacher and the students but the teachers' practice was poor; and the relationship between the students and the teacher was good but the teacher's expectations of the Pasifika students were low, and Pasifika learning was poor. In one vignette the relationship between the teacher and the students was one of trust and the teacher used teaching strategies that engaged and challenged the Pasifika students. In this classroom Pasifika learning was happening for a time. The study found that good Pasifika learning requires that the teacher must have all three of the following teaching strategies: allowing Pasifika students respect as a learner; being able to scaffold Pasifika learning at the right level and engaging their Pasifika students in active learning. The Pasifika student must have confidence and trust in their teacher to engage with the teacher in the active pursuit of learning. The classroom teacher must also have confidence in the Pasifika student's ability e.g., high expectations. If any of the parts described above are missing Pasifika learning is poor. The study demonstrates that the ways in which teachers fall into poor relationships and poor teaching practices can be related to their beliefs about Pasifika values and "Pasifika ways of learning".</p>

Application of modified Michaelis – Menten equations for determination of enzyme inducing and inhibiting drugs

Background Pharmacokinetics (PK) is the process of absorption, distribution, metabolism and elimination (ADME) of drugs. Some drugs undergo zero-order kinetics (ethyl alcohol), first order kinetics (piroxicam) and mixed order kinetics (ascorbic acid). Drugs that undergo Michaelis-Menten metabolism are characterized by either increased or decreased metabolism constant (Km) and maximum velocity (Vmax) of enzyme reaction. Hence literatures were searched with a view to translating in vitro-in vivo enzyme kinetics to pharmacokinetic/pharmacodynamic parameters for determination of enzyme inducing and inhibiting drugs, in order to achieve optimal clinical efficacy and safety. Methods A narrative review of retrospective secondary data on drugs, their metabolites, Vmax and Km, generated in the laboratory and clinical environments was adopted, using inclusion and exclusion criteria. Key word search strategy was applied, to assess databases of published articles on enzyme inducing and inhibiting drugs, that obey Michaelis-Menten kinetics. In vitro and in vivo kinetic parameters, such as concentration of substrate, rate of endogenous substrate production, cellular metabolic rate, initial velocity of metabolism, intrinsic clearance, percent saturation and unsaturation of the enzyme substrate, were calculated using original and modified formulas. Years and numbers of searched publications, types of equations and their applications were recorded. Results A total of fifty-six formulas both established and modified were applied in the present study. Findings have shown that theophylline, voriconazole, phenytoin, thiopental, fluorouracil, thyamine and thymidine are enzyme inducers whereas, mibefradil, metronidazole, isoniazid and puromicin are enzyme inhibitors. They are metabolized and eliminated according to Michaelis-Menten principle. The order could be mixed but may change to zero or first order, depending on drug concentration, frequency and route of drug administration. Conclusion Hence, pharmacokinetic-pharmacodynamic translation can be optimally achieved by incorporating, newly modified Michaelis-Menten equations into pharmacokinetic formulas for clinical efficacy and safety of the enzyme inducing and inhibiting therapeutic agents used in laboratory and clinical settings.