A QM/MM study of the catalytic mechanism of aspartate ammonia lyase

Aspartate ammonia lyase (Asp) is one of three types of ammonia lyases specific for aspartate or its derivatives as substrates, which catalyzes the reversible reaction of l-aspartate to yield fumarate and ammonia. In this paper, the catalytic mechanism of Asp has been studied by using combined quantum-mechanical/molecular-mechanical (QM/MM) approach. The calculation results indicate that the overall reaction only contains two elementary steps. The first step is the abstraction of C_β proton of l-aspartate by Ser318, which is calculated to be rate limiting. The second step is the cleavage of C_αN bond of l-aspartate to form fumarate and ammonia. Ser318 functions as the catalytic base, whereas His188 is a dispensable residue, but its protonation state can influence the active site structure and the existing form of leaving amino group, thereby influences the activity of the enzyme, which can well explain the pH dependence of enzymatic activity. Mutation of His188 to Ala only changes the active site structure and slightly elongates the distance of C_β proton of substrate with Ser318, causing the enzyme to remain significant but reduced activity.     

Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

H₂ production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H₂ production by fermentative bacteria by maximizing the number of electrons that are available for H₂. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O₂)-binding protein on oxic H₂ production in a closed batch system that was supplemented with glucose. The H₂ yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O₂ tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H₂ yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H₂ production and not by working as an O₂-scavenger during H₂ production. This new approach allowed more substrate to remain to be further utilized for the production of more H₂ from limited resources. We expect that VHb can be successfully engineered in potential aerobic H₂-producing microbial systems to enhance the overall H₂ production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H₂.     

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.     

Reassessment of treatments to retard browning of fresh‐cut Russet potato with emphasis on controlled atmospheres and low concentrations of bisulphite

The cultivar Pacific Russet with high browning susceptibility was used for most testing. Controlled atmospheres (0.3%, 3% and 21% O₂ in combination with 0%, 6% or 12% CO₂) and anti‐browning chemicals were studied in relation to quality retention and wound‐induced phenolic metabolism of fresh‐cut slices for up to 16 days at 5 °C. The 3% O₂+ 12% CO₂ atmosphere was most effective among those tested, and retarded increases in phenolics and phenylalanine ammonia lyase activity, but had only slight benefit on visual quality. A 1.25% ascorbic acid +1.25% citric acid treatment was ineffective, but when combined with 3% O₂+ 12% CO₂, it was comparable with 0.025% sodium bisulphite. Bisulphite concentrations from 0.05% to 0.25% provided similar effective control of discolouration. Bisulphite as low as 0.025% with 3% O₂+ 12% CO₂ resulted in a visual quality score at the limit of marketability after 8 days at 5 °C. Chemical treatments did not retard increases in phenolic concentrations or phenolic enzyme activities.     

放线菌果胶酶的分离纯化及酶学性质的研究

对放线菌产生的果胶酶进行了分离纯化，获得了电泳纯的组份Ｅ１。并对Ｅ１的最适作用ｐＨ、温度，ｐＨ、温度及贮存稳定性，分子量、等电点、ｋｍ、Ｖｍ、氨基酸组成、裂解酶活、水解酶活进行了研究。     

木薯醇腈酶部分酶学性质研究

对利用基因重组技术获得的木薯醇腈酶（HNL）的酶学特异性进行研究，确定了其部分酶学性质。重组木薯HNL的最适温度为50℃，最适pH值为5．5。重组木薯HNL在60℃条件下，保温30min后仍具有90％的酶活力，说明温度稳定性较好；在pH4．0～6．0的条件下，获得的相对酶活仍大于80％。     

Screening of phenylalanine ammonia lyase in plant tissues,and retention of activity during dehydration

BACKGROUND: Oral therapy with phenylalanine ammonia lyase (PAL), naturally encapsulated in plant cells, may provide a potential alternative treatment for hyperphenylalaninemic patients, including those with phenylketonuria. Therefore different sources of plant tissue were investigated for PAL activity. RESULTS: Enzyme activity was highest in grain seedlings, with maximal enzyme activity in 7‐day‐old red spring wheat (Triticum aestivum L.) seedlings. The PAL activities of leaves and roots/endosperm of wheat seedlings were 11.90 ± 2.64 and 6.48 ± 1.59 µmol h^?1 g^?1 dry weight respectively. Three PAL‐related polypeptides with molecular weights of 74, 83 and 103 kDa were identified in wheat seedling leaf tissues, while only the 74 kDa polypeptide was detected in root/endosperm tissues. Dehydration was investigated as a method of concentrating PAL in wheat seedlings. Freeze‐drying was found to retain the most PAL activity (>90% recovery on a dry weight basis) compared with air drying and vacuum microwave drying for both leaf and root/endosperm samples. CONCLUSION: This study has led to a better understanding of PAL activity and stability in plant tissues and provides the basis for developing a natural plant preparation as a dietary supplement for the treatment of hyperphenylalaninemia. Copyright © 2007 Society of Chemical Industry