L-氨基酸氧化酶的研究进展

L-氨基酸氧化酶(L-amino acid oxidase, LAAO)能特异性催化L-氨基酸氧化脱氨,生成α-酮酸、氨和H₂O₂。该酶分布较广,其中蛇毒源LAAO是该类酶中研究最为深入的一类,近年来,越来越多的非蛇毒源LAAO被发现和报道,现对蛇毒源和非蛇毒源LAAO的研究进展进行了综述。现有研究表明,不同物种来源的LAAO,其底物选择性、等电点、稳定性等理化性质不尽相同;虽对其结构的研究还较少,但现有的研究表明蛇毒源和非蛇毒源LAAO的结构都含有FAD结合结构域、底物结构域和螺旋结构域;研究已发现不同来源的LAAO体外具有多种不同的生物学功能,而这些生物学功能多数是由于其产物H₂O₂作用的结果;对LAAO异源表达的研究较少且都不甚成功,可能是由于其需要进行翻译后修饰。     

蛇毒L-氨基酸氧化酶酶学性质及对血小板功能的影响

L-氨基酸氧化酶(EC1·4·3·2)是一种黄素类蛋白酶,其酶活性为立体特异性催化L-氨基酸的氧化脱氨,生成α-酮酸,同时产生氨和过氧化氢。酶广泛存在于多种生物体种类中。蛇毒L-氨基酸氧化酶(LAAO)是该类酶中研究最为深入的一族,因其易于纯化而成为研究L-氨基酸氧化酶的酶学、结构生物学和药物学的有趣客体。近年来,越来越多的具有不同分子量、不同底物选择性的蛇毒LAAO被分离纯化,国内外文献越来越多的报道了蛇毒LAAO的特性,如准确的分子量、底物特性、与血小板的相互作用、诱发出血和凋亡。本文就蛇毒LAAO的性质及其对血小板功能的…     

生物催化氨基酸C—N裂解反应的研究进展

氨基酸脱氨酶能催化系列氨基酸C—N裂解反应生成对应的α 酮酸和氨,是代谢途径及生物催化的重要酶.综述了近年来催化氨基酸C—N裂解反应的5'磷酸吡哆醛介导的苏氨酸脱氨酶、黄素腺嘌呤二核苷酸介导的L氨基酸脱氨酶和L氨基酸氧化酶,以及这些关键酶应用于多酶级联反应中以生产α 羟基酸、α 酮酸、D氨基酸等精细化学品的研究进展.此...     

利用巨大芽孢杆菌酶系合成L-丙氨酸和L-缬氨酸

本文研究了利用巨大芽孢杆菌ATCC_(39118)酶系合成氨基酸,同时也研究了丙氨酸脱氢酶、缬氨酸脱氢酶及葡萄糖脱氢酶的提纯工艺。所获得的AlaDH、ValDHc和GlcDH的比活性分别为11.2u/mg,7.8u/mg和23.0u/mg。为了进一步探讨由α-酮酸酶法转化成氨基酸的最适条件,我们对以上三种酶的主要性质,包括稳定性,最适pH、动力学常数、底物专一性及底物和产物对酶的抑制作用等进行了测定。同时用粗酶提取液和纯酶进行了由丙酮酸合成L-丙氨酸,由α-酮异戍酸合成L-缬氨酸的批量实验,在转化中葡萄糖脱氢酶作为NADH的再生酶。结果粗酶提取液催化L-丙氨酸产量的克分子转化率为80％,而纯酶催化的克分子转化率增加到92％。L-缬氨酸产量的克分子转化率也类似(93％)。     

转氨酶催化不对称合成芳香族L-氨基酸

芳香族L-氨基酸是合成许多药物、农药、精细化学品和食品添加剂的重要手性砌块(Chiral buildingblocks)。利用酶催化具有高活性和高立体选择性的特点合成手性砌块是目前不对称合成领域重要的研究方向。通过对不同来源转氨酶的进化分析,选择分别源自原核生物大肠杆菌Escherichia coli和真核生物酿酒酵母Saccharomyces cerevisia中的两种具有代表性Ⅰ型芳香族转氨酶TyrB和Aro8,比较研究了两种转氨酶通过平衡逆转不对称氨化催化合成芳香族L-氨基酸的反应过程和催化效率。重组转氨酶TyrB和Aro8都能有效地合成天然芳香族氨基酸苯丙氨酸和酪氨酸以及非天然氨基酸苯甘氨酸。手性HPLC分析表明,合成的氨基酸都是L-构型的,e.e值等于100%。L-丙氨酸是适宜的氨基供体,转氨酶TyrB和Aro8都不能利用D-型氨基酸作为氨基供体。反应体系中氨基供体L-丙氨酸和氨基受体芳香族α-酮酸的最适摩尔比为4∶1。底物芳香族α-酮酸分子结构中芳香环上的取代基以及脂肪酸碳链部分的长度都对酶催化的转氨效率有显著的影响。在制备规模试验中,TyrB催化不对称转氨反应合成L-苯甘氨酸、L-苯丙氨酸和L-酪氨酸的比生产速率为0.28 g/(g.h)、0.31 g/(g.h)和0.60 g/(g.h),Aro8催化上述反应的比生产速率分别为0.61 g/(g.h)、0.48 g/(g.h)和0.59 g/(g.h)。研究结果对利用转氨酶通过平衡逆转不对称催化合成芳香族L-氨基酸的工业化应用具有指导意义。     

蛇毒L-氨基酸氧化酶抗肿瘤作用研究进展

L-氨基酸氧化酶（LAAO）广泛地存在于各种蛇毒中。近年来许多文献报道了蛇毒L-氨基酸氧化酶具有与血小板相互作用、细胞毒性及诱导细胞凋亡作用,还报道它们具有出血或溶血活性、引起水肿及抗细菌、抗艾滋病病毒的特性。本文就其抗肿瘤方面的研究进行综述,介绍蛇毒L-氨基酸氧化酶的抑瘤机制、理化性质及国内外研究现状和应用前景。     

海因酶的催化特性及其反应机理*

目前,已发现多种水解海因类的酰胺酶,如D-海因酶、二氢嘧啶酶、尿囊素酶、亚酰胺酶等海因酶。海因酶具有底物依赖的对映体选择性。在金属离子的参与下,它催化海因、5’-单替代海因及其衍生物发生水解。海因酶是有重要应用价值的工业酶,被广泛应用于生产具光学活性的D-和L-型氨基酸。综述了海因酶的催化特性、反应机理及应用前景。     

苏氨酸醛缩酶的结构与功能及其在药物合成中的应用

β-羟基-α-氨基酸(β-hydroxy-α-amnio acids,HAAs)是一类广泛应用于制药工业的重要手性中间体。由于其含有双手性中心(C_α和C_β),探索其严格立体选择性的生物合成方法备受关注。苏氨酸醛缩酶(threonine aldolase,TA)可在温和条件下催化不同类型的醛与氨基酸缩合构筑丰富的HAAs产物库,显示了工业应用潜力。由于目前表征的TA普遍存在对C_β立体选择性不严格、活性较低以及催化机制不清晰等问题,为其在HAAs合成中的应用带来了挑战。本文综述了TA在新酶挖掘、结构与催化机理解析、蛋白质工程以及合成应用等方面的研究进展,为推动酶催化绿色、高效合成手性药物提供参考。     

α-氨基酸酯酰基转移酶表达纯化、酶学性质及其催化应用

α-氨基酸酯酰基转移酶(α-amino acid ester acyltransferase,AET)能够催化底物L-丙氨酸甲酯盐酸盐、L-谷氨酰胺合成L-丙氨酰-L-谷氨酰胺(L-alanyl-L-glutamine,丙谷二肽)。利用重组大肠杆菌saet-QC01表达α-氨基酸酯酰基转移酶,对其表达条件进行了优化,通过Ni-NTA亲和层析法分离纯化重组蛋白,并对其酶学性质、催化应用进行了研究。适合酶表达的诱导条件:温度20℃,诱导阶段(OD_(600)=2.0-2.5),IPTG浓度0.6 mmol/L,诱导时间12 h。α-氨基酸酯酰基转移酶的最适反应温度27℃,最适pH 8.5,在pH 7.0-8.0很稳定,在酸性条件下相对稳定,低浓度的Co~(2+)、低浓度的EDTA对酶活有促进作用。在底物浓度丙氨酸甲酯盐酸盐600 mmol/L、谷氨酰胺480 mmol/L,丙谷二肽的产量达到78.2 g/L,生产速率达到1.955 g/(L·min),转化率达到75.0%。α-氨基酸酯酰基转移酶具有良好的酸碱耐受性,催化效率高的优良特性,在工业生产中具有较好的应用潜力。     

植物抗坏血酸过氧化物酶的表达调控以及对非生物胁迫的耐受作用

抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)属于I型血红素过氧化物酶, 它催化H2O2依赖的L-抗坏血酸氧化作用, 对抗坏血酸表现出高度的专一性。植物APX基因家族由4个亚家族组成, 分别为细胞质、叶绿体、线粒体和过氧化物酶体基因亚家族, 每个亚家族中又含有不同的APX同工酶。作为植物抗坏血酸-谷胱甘肽循环中的一个关键组分, APX在细胞H₂O₂代谢过程中起着至关重要的作用。研究表明植物APX是氧化还原信号系统中调节细胞水平H₂O₂非常重要的一种酶, APX同工酶的表达机制非常复杂, 细胞质APX受多种信号调节表达, 两种叶绿体APX通过选择性剪接进行组织特异性调节。通过调控产生的APX可调节细胞中的氧化还原信号, 进而提高植物对非生物胁迫的耐受性。文章综述了植物APX的催化机制、表达调控机理以及响应植物非生物逆境胁迫的重要作用。     

l-Amino acid oxidases from microbial sources: types,properties, functions,and applications

l-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of l-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotide-containing homodimeric proteins. l-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. Because expressing LAAOs as recombinant proteins in heterologous hosts is difficult, their biotechnological applications have not been thoroughly advanced. LAAOs are thought to contribute to amino acid catabolism, enhance iron acquisition, display antimicrobial activity, and catalyze keto acid production, among other roles. Here, we review the types, properties, structures, biological functions, heterologous expression, and applications of LAAOs obtained from microbial sources. We expect this review to increase interest in LAAO studies.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

氨基酸脱氢酶的催化机理、分子改造及合成应用

氨基酸脱氢酶催化可逆的氨基酸氧化脱氨和酮酸的不对称还原胺化反应,热力学上反应平衡倾向于生成氨基酸方向,从原子经济学和对环境影响的角度来看,是具有极大优势的氨基酸合成方法之一。本文将主要阐述近年来在?-氨基酸脱氢酶催化机理、分子改造和合成应用方面的研究进展。     

Cloning of L-amino acid deaminase gene from Proteus vulgaris

The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.     

Structural insights into selectivity and cofactor binding in snake venom L-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH₃) and hydrogen peroxide (H₂O₂). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1 Å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62–71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode.     

Screening and development of enzymes for determination and transformation of amino acids

ABSTRACT

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. Here, I review the discovery of l-Phe dehydrogenase and its practical use in the diagnosis of phenylketonuria in more than 5,400,000 neonates over two decades in Japan. Screening and uses of other selective enzymes for micro-determination of amino acids have also been discussed. In addition, novel enzymatic assays with the systematic use of known enzymes, including assays based on a pyrophosphate detection system using pyrophosphate dikinase for a variety of l-amino acids with amino-acyl-tRNA synthetase have been reviewed. Finally, I review the substrate specificities of a few amino acid-metabolizing enzymes that have been altered, using protein engineering techniques, mainly for production of useful chemicals, thus enabling the wider use of natural enzymes.     

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.