Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases(UGTs)mediate the glycosylation of plant metabolites,thereby altering their physicochemical properties and bioactivities.Plants possess numerous UGT genes,with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics,but the biological function and molecular basis of these phenomena are not fully understood.The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition(Ki)at 4 mM scopoletin,whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 mM.We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs.A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reducedscopoletin substrate inhibition,whereas its monolignolgly cosylation activity was almost unaffected.Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation,leading to enhanced promiscuity,which was accompanied by substrate inhibition.Studies of 3D structures identified open and closed UGT conformers,allowing visualization of the dynamics of conformational changes that occur during catalysis.Previously postulated substrate access tunnels likely serve as drainage channels.The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release.Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions.The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Microbial Degradation of Quinoline: Kinetics Study With Burkholderia picekttii

Objective To investigate the kinetics of quinoline biodegradation by Burkholderia pickttii, a Gram negative rod-shaped aerobe, isolated in our laboratory. Methods HPLC (Hewlett-Packard model 5050 with an UV detector) was used for the analysis of quinoline concentration. GC/MS method was used to identify the intermediate metabolites of quinoline degradation. Results The biodegradation of quinoline was inhibited by quinoline at a high concentration, and the degradation process could be described by the Haldane model. The kinetic parameters based on Haldane substrate inhibition were evaluated. The values were v = 0.44 h-1,Ks=166.7 mg/L, Ki= 650 mg/L, respectively. The quinoline concentration to avoid substrate inhibition was inferred theoretically and determined to be 329 mg/L. Conclusion The biodegradation of quinoline conforms to the Haldane inhibition model and the main intermediate metabolite of quinoline biodegradation is 2-hydroxy-quinoline.

Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.

Impact of undissociated volatile fatty acids on acidogenesis in a two-phase anaerobic system

This study investigated the degradation and production of volatile fatty acids（VFAs）in the acidogenic phase reactor of a two-phase anaerobic system.20 mmol/L bromoethanesulfonic acid（BESA）was used to inhibit acidogenic methanogens（which were present in the acidogenic phase reactor）from degrading VFAs.The impact of undissociated volatile fatty acids（un VFAs）on＂net＂VFAs production in the acidogenic phase reactor was then evaluated,with the exclusion of concurrent VFAs degradation.＂Net＂VFAs production from glucose degradation was partially inhibited at high un VFAs concentrations,with 59%,37% and 60% reduction in production rates at 2190 mg chemical oxygen demand（COD）/L undissociated acetic acid（un HAc）,2130 mg COD/L undissociated propionic acid（un HPr）and 2280 mg COD/L undissociated n-butyric acid（un HBu）,respectively.The profile of VFAs produced further indicated that while an un VFA can primarily affect its own formation,there were also un VFAs that affected the formation of other VFAs.