Novel biosensors based on optimized glycine oxidase

Abstract

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 lM). IntroductionGlycine is the smallest and the only nonchiral natural a-amino acid used for protein biosynthesis. In addition to such a structural role, it possesses further physiological functions: it is an important biosynthetic precursor (i.e. it is used for de novo synthesis of porphyrins and purines) and it acts as a neurotransmitter in the central nervous system. Glycine is a coagonist, together with D-serine, of glutamate at glutamatergic N-methyl-Daspartate receptors (NMDARs) in the forebrain [1], thus serving both inhibitory and excitatory functions [2]. Due to this role, glycine is probably involved in the pathophysiology of schizophrenia and other neurological diseases. For example, a correlation exists between low levels of glycine and catatonia (a psychiatric disease), and the perturbation of the glycine deportation system is associated with ifosfamide encephalopathy and congenital nonketotic hyperglycinemia [2].Sarcosine, 2-(methylamino)acetic acid or N-methylglycine, is a natural, small, endogenous amino acid present in low concentrations in the blood and in urine. It has been proposed to play a role in the progression of prostate cancer, and assessing sarcosine concentrations in body fluids was suggested as a novel biomarker for prostate cancer [3]. With the final aim of developing a biosensor to assay glycine and sarcosine in biological fluids, we engineered glycine oxidase from Bacillus subtilis (GO) (EC 1.4.3.19) to improve its kinetic efficiency on these two compounds. GO is a homotetrameric flavoenzyme that contains one molecule of noncovalently bound FAD per 47 kDa protein monomer [4,5]. Purified recomb...

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus

“…1 A and B). These responses were only reduced by 35.5 ± 11.5% when endogenous glycine was degraded with a novel highly active variant of Bacillus subtilis glycine oxidase (BsGO) (0.2 U/mL) (15) [F (1,140) = 29.37; P < 0.0001; n = 7] (Fig. 1B).…”

Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria

“…This protein shares a similar substrate range and structure with the glycine oxidase from B. subtilis . Regarding applications, glycine oxidase can be used as a biosensor for glycine in biological samples [ 82 ], and in the degradation of the herbicide glyphosate [ 83 , 84 ]. Flavoproteins with sarcosine oxidase activity (SOX, EC 1.5.3.1), like the one from Bacillus sp.…”

A comprehensive practical laboratory course on protein engineering: Evolution of a glycine oxidase variant active on the herbicide glyphosate

“…No active clones are obtained from the screening of the library at position 302, thus confirming an important role of this residue in substrate binding . Moreover, for the libraries at positions 95, 241, and 329, none of the clones should show an activity on glycine higher than the wild‐type enzyme .…”

“…On the fourth day, each group screens a library corresponding to a single position, session 4. The day before, the technical assistant picks ~ 90 single colonies (and different clones as controls, see below) and grows recombinant E. coli cultures (1 mL) overnight to saturation at 37 °C in a 2‐mL deep‐well plate in LB medium (after adding 100 μg/mL Am and 34 μg/mL Chl) . In the morning, 0.5 m M IPTG are added to the cell cultures, which are incubated at 30 °C for 5 hours.…”

“…In 2018 the Nobel prize for Chemistry was awarded with one half to Frances H. Arnold for the “directed evolution of enzymes”. The engineering of proteins represents a powerful approach by which proteins (and frequently enzymes) are generated that are well suited for applications in different fields as biocatalysts, therapeutic agents, and biosensors . Knowledge of basic skills in protein engineering is mandatory for future biotechnologists.…”