内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

生物转化和手性拆分技术制备D-氨基酸研究进展

在自然界中D-氨基酸相对稀少,被冠以“非天然”氨基酸之称,但D-氨基酸在医药、农药和食品等的组成中起着重要的作用,特别是它们已被用于合成!-内酰胺类抗生素和生理活性肽。综述了通过生物转化和手性拆分技术制备D-氨基酸新进展,希望能为相关研究者和有关企业提供有益的参考。     

海因酶研究新进展

海因酶在生产手性药物中间体D-氨基酸上具有广泛的应用价值。全面综述了海因酶的研究历史、分类、进化和酶学性质;总结了产海因酶的茵种筛选和产酶条件、D-海因酶的纯化、微生物海因酶基因的克隆及其在大肠杆茵中的表达等技术;阐述了酶法合成D-(-)-对羟基苯甘氨酸的工艺条件。     

D型氨基酸氧化酶活性对于D-硝基精氨酸手性转化的影响

D-硝基精氨酸(D-NNA)可在大鼠体内发生手性转化生成其L型异构体,即L-NNA,后者可抑制一氧化氮合酶活性,减少一氧化氮生成,升高动脉血压.研究了D型氨基酸氧化酶(DAAO)在D-NNA手性转化中的作用及DAAO对不同(包括已报道在体内可发生手型转化的)D型氨基酸的选择活性.体内实验显示,DAAO的选择性抑制剂苯甲酸钠(400mg/kg)或肌酐(400mg/kg)均可在不同程度上抑制D-NNA升压作用,进一步研究发现,肾脏或肝脏DAAO酶液在外加DAAO后可提高D-NNA的手性转化约2倍,表明DAAO对于D-NNA在体内的手性转化是必需的.DAAO酶液对可在体内发生手性转化且转化率相似(30%～50%)的D型氨基酸(D-Phe,D-Leu和D-NNA)的选择性表现出显著差异(Kcat/Km相差可达约15倍左右),这从另一方面表明体内D-硝基精氨酸氧化是其发生手性转化的前提条件但非决定因素.     

氨基酸手性同一起源的新理论模型

氨基酸手性同一, 这一生命体系特有的行为, 可能伴随着密码子的起源而形成. 在此假设的前提下, 本文将带有P-N键的核苷-5′-磷酰化氨基酸化合物作为前生物时期的手性起源模型来探讨手性的选择. 在这个模型化合物中, 由于氨基酸手性原子的存在, 导致氨基酸侧链和核苷酸碱基之间出现最佳空间取向差异, 进而影响到分子内部不同官能基团之间的相互作用, 氨基酸的某种手性可能成为优势构型, 有利于该化合物的稳定. 为了验证这一氨基酸手性同一起源模 型, 本文从实验和理论上做了初步研究. 研究结果显示, 古老氨基酸的L型异构体优先被选择遵从立体/物理化学决定论, 而随后出现的氨基酸, 其手性的选择可能是手性继承的结果. 以上工作是氨基酸手性同一起源的一种猜测, 为进一步开展实验验证提供依据和思路.     

非天然氨基酸及非天然蛋白合成的研究进展

非天然氨基酸是天然氨基酸的衍生物,其具有重要的生理和药理功能.由非天然氨基酸合成的非天然蛋白同样具有特别的功能与活性.非天然氨基酸插入蛋白质中的研究为新型生物材料和蛋白质药物等的合成和应用提供了新的指导方向.基于此,本文综述了非天然氨基酸的生物合成方法,分析了非天然氨基酸生物合成目前存在的难点,总结了非天然氨基酸插入蛋...     

D—苯丙氨酸的不对称合成

本文以D-樟脑与甘氨酸持丁酯缩合的D-樟脑酮亚胺为手性合成子,通过不对称苄基化反应,再经酸水解,对映选择合成了D—苯丙氨酸,其光学纯度和对映体过量百分率达95%,这是一种合成光学纯氨基酸的新方法。     

对称性破缺与生命起源

对称性破缺是自然界的基本规律,手性均一为生命起源的必要条件。自然界中组成生命的蛋白质有２０种氨基酸（除甘氨酸无不对称碳原子外）全是Ｌ型,组成ＲＮＡ、ＤＮＡ中的核糖全是Ｄ型。近年研究发现：人体中Ｄ型氨基酸广泛存在。维持人体手性均一有赖于Ｄ－氨基酸氧化酶、Ｄ－天冬氨酸氧化酶的作用。本文对当前国际上最著名的两大学说－极化电子和手性分子相互作用与萨拉姆假说及国内外研究的最新进展,结合笔者科研组十几年来的实验研究和理论观点作较为全面的评述,并提出新的理论解释,解决长期争论的实验分歧。     

基于HNP三态模型及相对熵方法的蛋白质折叠研究

把20种氨基酸简化为3类:疏水氨基酸(hydrophobic,H)、亲水氨基酸(hydrophilic,P)及中性氨基酸(neutral,N),每个氨基酸简化为一个点,用其C!原子来代替.采用非格点模型,以相对熵作为优化函数,进行蛋白质三维结构预测.为了与基于相对熵方法的蛋白质设计工作进行统一,采用了新的接触强度函数.选用蛋白质数据库中的天然蛋白质作为测试靶蛋白,结果表明,采用该模型和方法取得了较好的结果,预测结构相对于天然结构的均方根偏差在0.30～0.70nm之间.该工作为基于相对熵及HNP模型的蛋白质设计研究打下了基础.     

普萘洛尔对映异构体诱导HUVEC细胞的蛋白质表达谱差异

手性药物只能通过严格的手性识别才能选择性地与特定生物大分子相互作用，在药动学、药效学等方面上表现出手性特征.以非选择性β肾上腺素能受体阻滞剂普萘洛尔（PRO）的对映异构体R(+)/S(-)-PRO为模型药物，分别作用于人脐静脉内皮细胞（HUVEC），提取全细胞蛋白质，经双向电泳、MALDI-TOF-MS、SWISSPROT数据库分析鉴定差异表达蛋白质；共筛选出22个差异表达蛋白质点，鉴定了HSP86、HSP84、GRP75、KLC18、KBTB2、TGM2、GBLP、GCNT2、RAB36、KLH34等10种蛋白质.研究表明，PRO对映异构体可引起广泛的基因表达改变，涉及信号分子、代谢酶、骨架蛋白、伴侣蛋白等，且具有显著的手性特征，这可能与PRO显著的手性生物学特征有紧密联系，但仍需开展进一步深入研究，以明确产生PRO手性生物学特征的多种途径和机制.蛋白质组学技术为深入了解药物的手性生物学特征及其作用机制提供了新的思路和策略，对手性药物开发和临床合理用药有着重要的意义.     

D-Amino Acids in Living Higher Organisms

The homochirality of biological amino acids (L-amino acids) andof the RNA/DNA backbone (D-ribose) might have become establishedbefore the origin of life. It has been considered that D-aminoacids and L-sugars were eliminated on the primitive Earth.Therefore, the presence and function of D-amino acids in livingorganisms have not been studied except for D-amino acids in thecell walls of microorganisms. However, D-amino acids wererecently found in various living higher organisms in the form offree amino acids, peptides, and proteins. Free D-aspartate andD-serine are present and may have important physiologicalfunctions in mammals. D-amino acids in peptides are well knownas opioid peptides and neuropeptides. In protein, D-aspartateresidues increase during aging. This review deals with recentadvances in the study of D-amino acids in higher organisms.     

A D-amino acid editing module coupled to the translational apparatus in archaea

We report the crystal structure of an archaea-specific editing domain of threonyl-tRNA synthetase that reveals a marked structural similarity to D-amino acid deacylases found in eubacteria and eukaryotes. The domain can bind D-amino acids despite a low sequence identity to other D-amino acid deacylases. These results together indicate the presence of these deacylases in all three kingdoms of life. This underlines an important role they may have played in enforcing homochirality during translation.     

d-Amino acids in molecular evolution in space – Absolute asymmetric photolysis and synthesis of amino acids by circularly polarized light

Living organisms on the Earth almost exclusively use l-amino acids for the molecular architecture of proteins. The biological occurrence of d-amino acids is rare, although their functions in various organisms are being gradually understood. A possible explanation for the origin of biomolecular homochirality is the delivery of enantioenriched molecules via extraterrestrial bodies, such as asteroids and comets on early Earth. For the asymmetric formation of amino acids and their precursor molecules in interstellar environments, the interaction with circularly polarized photons is considered to have played a potential role in causing chiral asymmetry. In this review, we summarize recent progress in the investigation of chirality transfer from chiral photons to amino acids involving the two major processes of asymmetric photolysis and asymmetric synthesis. We will discuss analytical data on cometary and meteoritic amino acids and their potential impact delivery to the early Earth. The ongoing and future ambitious space missions, Hayabusa2, OSIRIS-REx, ExoMars 2020, and MMX, are scheduled to provide new insights into the chirality of extraterrestrial organic molecules and their potential relation to the terrestrial homochirality. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.     

D-Amino acids in mammals and their diagnostic value

Substantial amounts of D-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine D-amino acids in mammalian tissues are reviewed, and the distribution of these D-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.     

Epimerization of Alanyl-Alanine Induced by γ-Rays Irradiation in Aqueous Solutions

Living organisms have homochiral L-amino acids in proteins and homochiral D-mononucleotides in nucleic acids. The chemical evolutionary process to protein homochirality has been discussed for many years. Although many scenarios have been proposed for homochirality in the monomeric compounds, homochirality in amino acids and mononucleotides does not always guarantee homochirality in polypeptides and polynucleotides. Integrated scenarios containing the pathways from monomer to polymer should be proposed because in the pathways oligomers and polymers as well as monomers racemize (or epimerize), degrade, and condense. This research addresses epimerization and degradation of dipeptides under γ-rays irradiation by a cobalt-60 (⁶⁰Co) radiation source. The different rate constants of epimerization between diastereomeric dipeptides in the research suggest that the potential pathway toward homochirality could be much more complex.     

Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli.

Certain D-amino acids, such as D-methionine and D-cystine, were incorporated into cells of Escherichia coli under conditions inhibiting protein and cell wall synthesis. Part of the radioactivity of D-14C-amino acids incorporated into the cells was found in the isolated cell wall peptidoglycan. A covalent linkage between the amino group of the D-amino acids and the peptidoglycan was presumed to be the main cause of the binding of the D-amino acids to peptidoglycan, because the amino group of the D-amino acids in the incorporation product was substituted. Whether the carboxyl terminus was substituted was unknown. The formation of the D-amino acid-peptidoglycan linkage was insensitive to beta-lactam antibiotics such as benzylpenicillin and ampicillin (500 micrograms/ml) and therefore was not due to the reaction of DD-transpeptidation which is involved in the biosynthesis of peptidoglycan. The D-amino acids also strongly inhibited the formation of peptidoglycan-bound lipoprotein in the E. coli cells. The results may suggest the correlation between binding of D-amino acid to peptidoglycan and inhibition of formation of the bound form of lipoprotein.     

A Model of Proto-Anti-Codon RNA Enzymes Requiring l-Amino Acid Homochirality

All living organisms encode the 20 natural amino acid units of polypeptides using a universal scheme of triplet nucleotide "codons". Disparate features of this codon scheme are potentially informative of early molecular evolution: (i) the absence of any codons for D-amino acids; (ii) the odd combination of alternate codon patterns for some amino acids; (iii) the confinement of synonymous positions to a codon's third nucleotide; (iv) the use of 20 specific amino acids rather than a number closer to the full coding potential of 64; and (v) the evolutionary relationship of patterns in stop codons to amino acid codons. Here I propose a model for an ancestral proto-anti-codon RNA (pacRNA) auto-aminoacylation system and show that pacRNAs would naturally manifest features of the codon table. I show that pacRNAs could implement all the steps for auto-aminoacylation: amino acid coordination, intermediate activation of the amino acid by the 5'-end of the pacRNA, and 3'-aminoacylation of the pacRNA. The anti-codon cradles of pacRNAs would have been able to recognize and coordinate only a small number of L-amino acids via hydrogen bonding. A need for proper spatial coordination would have limited the number of chargeable amino acids for all anti-codon sequences, in addition to making some anti-codon sequences unsuitable. Thus, the pacRNA model implies that the idiosyncrasies of the anti-codon table and L-amino acid homochirality co-evolved during a single evolutionary period. These results further imply that early life consisted of an aminoacylated RNA world with a richer enzymatic potential than ribonucleotides alone.     

The significance of D-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps

Background

D-amino acids are far less abundant in nature than L-amino acids. Both L- and D-amino acids enter soil from different sources including plant, animal and microbial biomass, antibiotics, faeces and synthetic insecticides. Moreover, D-amino acids appear in soil due to abiotic or biotic racemization of L-amino acids. Both L- and D-amino acids occur as bound in soil organic matter and as “free“ amino acids dissolved in soil solution or exchangeably bound to soil colloids. D-amino acids are mineralized at slower rates compared to the corresponding L-enantiomers. Plants have a capacity to directly take up “free“ D-amino acids by their roots but their ability to utilize them is low and thus D-amino acids inhibit plant growth.

Scope

The aim of this work is to review current knowledge on D-amino acids in soil and their utilization by soil microorganisms and plants, and to identify critical knowledge gaps and directions for future research.

Conclusion

Assessment of “free“ D-amino acids in soils is currently complicated due to the lack of appropriate extraction procedures. This information is necessary for consequent experimental determination of their significance for crop production and growth of plants in different types of managed and unmanaged ecosystems. Hypotheses on occurrence of “free“ D-amino acids in soil are presented in this review.     

Factors controlling the level and determination of D-amino acids in the urine and plasma of laboratory rodents

Summary Unambiguous methodologies were developed for the accurate and reproducible determination of specific D-amino acids in the physiological fluids of common laboratory rodents. Depending on the strain of rodent and the type of amino acid examined, excreted D-amino acids ranged from the low percent levels to over 40 percent of the total specific amino acid level. Relative plasma levels tended to be considerably lower, typically an order of magnitude less. A number of factors were found to alter the relative amounts of excreted D-amino acids. This included: diet, age, pregnancy, advanced cancer, and antibiotics. The two factors that seemed to result in substantially lower levels of excreted D-amino acids were fasting and young age. Pregnancy was the only factor that consistently resulted in higher relative D-amino acid excretion. Much of the observed data are believed to be related to the efficiency with which the kidney reabsorbs L-amino acids. No claims are made as to the meaning and/or importance of free D-amino acids in regards to pathology, age, clinical usefulness and so forth. However, a knowledge of normal D-amino acid levels and dynamics is necessary before it is possible to identify perturbations caused by either natural or pathological conditions. The techniques are now available that should allow these topics to be addressed properly.On leave from Kyungpook National University in Korea.On leave from Institute of Physical Sciences, Polish Academy of Sciences.     

Second lytic target of beta-lactam compounds that have a terminal D-amino acid residue

A new biochemical mechanism of lysing bacterial cells by treatment with certain beta-lactam compounds that possess a terminal D-amino acid moiety in their side chain was demonstrated. The two functions of the molecule, the beta-lactam and terminal D-amino acid moiety, are both involved in the activity of lysing gram-negative bacteria, which is characterized by very rapid lysis of the cells in the first few hours after their contact with the compound. This mechanism was proved by studies on one such compound, named MT-141, which contains a terminal D-cysteine moiety with free amino and carboxyl groups in the 7 beta-side chain of the 7 alpha-methoxy-cephalosporin skeleton. This compound bound to the cell-wall peptidoglycan of Escherichia coli through the D-amino group of its terminal D-amino acid moiety and this seemed to cause rapid cell lysis. Both activities, of binding to peptidoglycan and of causing rapid cell lysis, were inhibited by certain D-amino acids, but not by L-amino acids. Mutants were isolated that had simultaneously gained decreased sensitivity to this kind of beta-lactam compound and supersensitivity to globomycin, an inhibitor of formation of lipoproteins which function in linking the peptidoglycan to the outer membrane. These results suggest that binding of the terminal D-amino acid moiety of the beta-lactam compound to peptidoglycan somehow influences formation of the linkage between the outer membrane and the peptidoglycan and consequently enhances the cell lytic activity of the beta-lactam portion of the molecule.