Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization.

Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size.

Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach.

Recently, the program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. We explored the utility of this program further and applied it for engineering of the aldoxime dehydratase from Bacillus sp. OxB-1. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. In part, this activity increase can be rationalised by an improved heme incorporation of the variants. For example, a single mutation gave a 1.8 fold increased enzymatic activity and 30% improved absolute heme incorporation.

Process properties of an l-amino acid oxidase from Hebeloma cylindrosporum for the synthesis of phenylpyruvic acid from l-phenylalanine.

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases.

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high-volume, low-price sectors, such as polymers, to low-volume, high-price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas-phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a "synthetic platform technology". Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2-arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.

Cyanide-Free and Broadly Applicable Enantioselective Synthetic Platform for Chiral Nitriles through a Biocatalytic Approach.

A cyanide-free platform technology for the synthesis of chiral nitriles by biocatalytic enantioselective dehydration of a wide range of aldoximes is reported. The nitriles were obtained with high enantiomeric excess of >90 % ee (and up to 99 % ee) in many cases, and a "privileged substrate structure" with respect to high enantioselectivity was identified. Furthermore, a surprising phenomenon was observed for the enantiospecificity that is usually not observed in enzyme catalysis. Depending on whether the E or Z isomer of the racemic aldoxime substrate was employed, one or the other enantiomer of the corresponding nitrile was formed preferentially with the same enzyme.