As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-...As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate(OPBE)with high R-enantiose-lectivity.This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis.Using this strategy,4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets,generating variant Mu27 with 99%conversion and 98%(R)ee value at substrate loading of up to 500 mmol/L.MD simu-lations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key rea-sons for the excellent R-enantioselectivity of Mu27 towards OPBE.The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.展开更多
The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a...The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a negetive correlation between the age of fetus and activity of AR(r=-0.810 1,0.05>P>0.01).展开更多
Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA...Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.展开更多
NADP(H) dependent retinol dehydrogenase/reductase (NRDR) was an important retinoic acid synthase, which was first purified from rabbit liver in 1997. In order to study the function of the NRDR gene,the full length cDN...NADP(H) dependent retinol dehydrogenase/reductase (NRDR) was an important retinoic acid synthase, which was first purified from rabbit liver in 1997. In order to study the function of the NRDR gene,the full length cDNA of bovine NRDR was cloned. According to the conserved sequences of human, mouse and rabbit NRDR cDNA, a pair of primers was designed to amplify a 294 bp DNA fragment of bovine liver NRDR, and then the full length of NRDR cDNA (AF487454) was cloned by using 3′ RACE and 5′ RACE. All the cloned NRDR proteins consist of 260 amino acid residues and showed high identity among them. The tri peptide of human, mouse and rabbit NRDR C end was SRL and that of bovine NRDR C end was SHL, but both were considered to be peroxisomal target signal 1 (PTS1). RT PCR demonstrated that NRDR gene was expressed in liver, heart, lung, kidney, stomach and intestine, and was not found in pancreas, muscle, artery and skin. The full length bovine NRDR cDNA has been successfully cloned and the sequence was analyzed. It provided a reliable foundation to investigate the biological function of this protein.展开更多
基金This study is financially supported by the National Key Research and Development Program of China(No.2018YFA0901800 to H.Yu and L.Ye)Zhejiang Provincial Natural Science Foundation of China(No.LY18B060001 to L.Ye and No.LZ20B060002 to H.Yu)Natural Science Foundation of China(No.21776244 to H.Yu).We are grateful to the constructive suggestions from academician Fen-Er Chen(Department of Chemistry,Fudan University,Shanghai,China)and the support of Discovery Studio software from Fuzhou University platform.
文摘As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate(OPBE)with high R-enantiose-lectivity.This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis.Using this strategy,4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets,generating variant Mu27 with 99%conversion and 98%(R)ee value at substrate loading of up to 500 mmol/L.MD simu-lations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key rea-sons for the excellent R-enantioselectivity of Mu27 towards OPBE.The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.
文摘The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a negetive correlation between the age of fetus and activity of AR(r=-0.810 1,0.05>P>0.01).
基金supported by grants from China Na-tional Basic Research Program (NO. 2004CB117302)National Natural Science Foundation of China (No.30470171)the Sigrid Jusélius Foundation Finland and the Academy of Finland
文摘Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.
文摘NADP(H) dependent retinol dehydrogenase/reductase (NRDR) was an important retinoic acid synthase, which was first purified from rabbit liver in 1997. In order to study the function of the NRDR gene,the full length cDNA of bovine NRDR was cloned. According to the conserved sequences of human, mouse and rabbit NRDR cDNA, a pair of primers was designed to amplify a 294 bp DNA fragment of bovine liver NRDR, and then the full length of NRDR cDNA (AF487454) was cloned by using 3′ RACE and 5′ RACE. All the cloned NRDR proteins consist of 260 amino acid residues and showed high identity among them. The tri peptide of human, mouse and rabbit NRDR C end was SRL and that of bovine NRDR C end was SHL, but both were considered to be peroxisomal target signal 1 (PTS1). RT PCR demonstrated that NRDR gene was expressed in liver, heart, lung, kidney, stomach and intestine, and was not found in pancreas, muscle, artery and skin. The full length bovine NRDR cDNA has been successfully cloned and the sequence was analyzed. It provided a reliable foundation to investigate the biological function of this protein.
