Sequence alignment of Nit6803 from Syechocystis sp. PCC6803 with other nitrilases

Secondary structure elements are drawn on the basis of structures of Nit6803 and shown at the top of the aligned sequences. b-Sheets are shown as arrows in yellow, whereas a-helices are shown as bars in red. Residues involved in enzymatic catalysis are indicated are highlighted in red rectangle, whereas the proposed key residue involved in substrate preference is highlighted in blue rectangle. Nit6803, PaNit, PH0642, DNCAase and RrNit indicate Syechocystis sp. PCC6803 nitrilase (GI: 16331918), hyperthermophilic nitrilase (GI: 14521598), Pyrococcus horikoshii hypothetical protein (GI: 14590532), N-carbamoyl-D-amino-acid amidohydrolase (GI: 34921541) and Rhodococcus rhodochrous ATCC 33278 nitrilase (GI: 417384).

 from this paper by Yuan, Wei and co-workers.

A crystal structure of nitrilase Nit6803 from Syechocystis sp. PCC6803

PDB: 3WUY_A

>gi|742261201|pdb|3WUY|A Chain A, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

PDB: 3WUY_B

>gi|742261202|pdb|3WUY|B Chain B, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

Two publications on the nitrilase from Sphingomonas wittichii RW1

Sphingomonas wittichii RW1 is a bacterium which has been shown to have the ability to degrade polychlorinated dioxins, and hence has had its genome fully sequenced. It has two different nitrilases in its genome.

YP_001264656.1

YP_001261492.1

Two papers just released with the corresponding authors Er-Zheng Su and Dong-Zhi Wei report on the nitrilase with the accession code YP_001264656. These authors and enzyme have previously been mentioned on this blog- I complained there was no information on the enzyme discovery... here it is!

The first entitled “Cloning, Overexpression, andCharacterization of a High Enantioselective Nitrilase from Sphingomonaswittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine” and published in Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology describes the construction of a mini-library of nitrilase enzymes chosen by the similarity to a nitrilase from Pseudomonas fluorescens EBC191 (GenBank accession no. AAW79573) which has been shown to be highly activity toward phenylglycinonitrile. The nitrilase from Sphingomonas wittichii RW1 was shown to be most promising nitrilase to achieve chiral synthesis of R-phenylglycine in a kinetic resolution mode.

The second entitled “Efficient asymmetric synthesis ofD-N-formyl-phenylglycine via cross-linked nitrilase aggregates catalyzeddynamic kinetic resolution” and published in Catalysis Communications describes the preparation of cross-linked enzyme aggregates (CLEAs) of nitrilase from Sphingomonas wittichii RW1, and then its use to perform dynamic kinetic resolution to make D-N-formyl-phenylglycine. The paper also demonstrates that the CLEAs have improved stability over non-immobilized enzyme and are reuseable up to six times whilst retaining 70% initial activity.

Dynamic Kinetic Resolution in alpha aminonitrile hydrolysis

The use of an equilibrium between enantiomers of your starting material to enable hydrolysis of more than 50% of a racemic mixture has been a point of keen interest in nitrilase research. The hurdle to it becoming widespread  has tended to be that the pH at which racemization occurs at a useful rate tends not to be one that your standard biocatalyst is happy operating at. A newly accepted manuscript into Tetrahedron Letters called “High yield synthesis of D-phenylglycine and its derivatives bynitrilase mediated dynamic kinetic resolution in aqueous-1-octanol biphasicsystem” by Jian Qiua, Erzheng Su, Wei Wang, and Dongzhi Wei is a useful addition to this literature. They use a nitrilase (after citing unpublished data on its enantioselectivity… why unpublished? It looks an interesting nitrilase!) from Sphingomonas wittichii RW1 to get DKR in a biphasic system (buffer/octanol).

Scaling up a nitrilase based hydrolysis

Efficient Production of (R)-o-Chloromandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315 by Dongzhi Wei and co-workers is published in Organic Process Research and Development at  DOI:10.1021/op400174a.  It being OPRD and from a group working in a Laboratory of Bioreactor Engineering, it’s going to be an interesting read on doing a nitrilase reaction on the larger scale.  We are currently working on scaling up a different nitrilase-based reaction currently from bench scale to the “too heavy to use normal glassware” scale so it’s good to see how others do it.

 

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