Masanori Kohmura

Complete Amino Acid Sequence of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.

SYNTHESIS AND CHARACTERIZATION OF THE SWEET PROTEIN BRAZZEIN

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless.

Structure of an Enantiomeric Protein, D-Monellin at 1.8 A Resolution.

The D-enantiomer of a potently sweet protein, monellin, has been crystallized and analyzed by X-ray crystallography at 1.8 A resolut ion. Two crystal forms (I and II) appeared under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin. There are four molecules per asymmetric unit in crystal form I and one in crystal form II. Crystal form I is not reproducible and is equivalent to that of monoclinic L-monellin. Intermonomer contacts in crystal form II are very different from those found in natural L-monellin crystals. The backbone trace of D-monellin resembles very closely the mirror image of that of L-monellin, but the N- and C-terminus backbones as well as several side-chain conformations of D-monellin are different from those of natural L-monellin. Most of these apparent differences may be attributable to the crystal packing differences.

ASSIGNMENT OF THE DISULFIDE BONDS IN THE SWEET PROTEIN BRAZZEIN

The thermostable sweet protein brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds, the location of which is unknown. We found that brazzein resists enzymatic hydrolysis at enzyme/substrate ratios (w/w) of 1:100‐1:10 at 35–40°C for 24–48 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5. for 6 h and at 50°C. The disulfide bonds were determined, by a combination of mass spectrometric analysis and amino acid sequencing of cystine‐containing peptides, to be between Cys4‐Cys52, Cys16‐Cys37, Cys22‐Cys47, and Cys26‐Cys49. These disulfide bonds contribute to its thermostability.

Chemical synthesis and characterization of the sweet protein mabinlin II

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste.

Structure and dynamic studies by NMR of the potent sweet protein monellin and a non-sweet analog: Evidence on the importance of residue AspB7 for sweet taste

Monellin, an intensely sweet protein and a non‐sweet analog in which the AspB7 in monellin has been replaced with AbuB7 were studied by NMR. The results of our investigations show that the 3‐dimensional structure of these two proteins are very similar indicating that the lack of the β‐carboxyl group in the AbuB7 analog is responsible for the loss of sweet potency. Selectively labeled monellin was prepared by solid‐phase peptide synthesis by incorporating 15N‐labeled amino acids into 10 key positions including AspB7. The internal mobility of these 10 key residues in monellin was estimated by the method of model‐free analyses and our NMR studies show that AspB7 is the most flexible of these 10 residues. The flexibility of the AspB7 side chain may be important for receptor binding.

Structure­taste relationships of the sweet protein monellin

Structuretaste relationship studies were carried out by chemically synthesizing monellin and its analogs. Replacement of the AspB7 by l-2-aminobutyric acid, Gly and d-Asp resulted in complete loss of sweetness. Replacement of IleB6 and IleB8 by different amino acids resulted in a significant decrease of sweetness, or complete loss of sweetness. Comparison of short- and long-range nuclear Overhauser effects (NOEs) and chemical shifts between monellin and [AbuB7]monellin showed no marked differences except for the region of the AspB7. Thus, the complete loss of sweetness in [AbuB7]monellin is caused by the lack of free β-carboxyl group in the AspB7 and not by a result of major disruption in the overall 3-dimensional structure. These results suggested that the free β-carboxyl group of the AspB7 would possibly bind to the receptor site through ionic bonding and trigger the sensation of intense sweet taste, and IleB6 and/or IleB8 would be involved in the hydrophobic interaction with the receptor site. Selectively labeled monellin was synthesized by the solid-phase method by incorporating 15N-labeled amino acids into 10 key residues including AspB7. Relaxation analysis shows that AspB7 is the most flexible of these 10 residues. The flexibility of the active site may be important for receptor binding.

Solid-Phase Synthesis and Crystallization of Monellin, an Intensely Sweet Protein

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of the A and B chains. The A and B chains corresponding to one of the reported monellin structures were synthesized by the stepwise solid-phase method using the Fmoc strategy in overall yields of 14.1% and 5.6%, respectively. The characterization of the synthetic peptides by HPLC, FAB-MS, amino acid analysis and sequencing fully supported the expected structures. The individual synthetic A and B chains were not sweet. Combination of the two chains, and subsequent HPLC purification gave monellin in a yield of 53.9%. The synthetic monellin had a distinct, lingering sweet taste (4000 times sweeter than sucrose) and was crystallized by a vapor diffusion method. The synthetic product was identical to natural monellin by HPLC, but not by tryptic mapping. These results indicate that the reported structure for monellin differs slightly from that of natural monellin.

Crystallization and preliminary X-ray analysis of d-monellin

D-Monellin is a chemically synthesized protein composed of all D-amino acids. It has an amino-acid sequence identical to L-monellin, a natural protein with potent sweetness. Two crystal forms of D-monellin were obtained. Both crystals were grown under conditions similiar to those used to crystallize natural L-monellin. Crystal form I has similar, but not identical, cell parameters to natural L-monellin and diffracts to 2.7 A resolution. Crystal form II is very different and diffracts to 1.7 A resolution using synchrotron radiation.

Human–Environment Interactions – Taste

Humans have evolved to consume natural products in their environment and have acquired a sense of taste. Our ability to taste bitter and sour evolved to identify potentially dangerous food. On the contrary, the ability to recognize a sweet taste developed to identify an energy source, while a salty taste is a signal of minerals. As time progressed, humans began to use bitter, pungent, and even astringent tastes that were initially considered to be unpleasant. This may have been because we became aware that such substances were effective at improving health or even treating disease. Human food culture progressed further with the development of cooking methods that use spices and more dramatically through the enjoyment of fermentation products. There has been no recent comprehensive review on the chemistry of taste written from the perspective of natural products chemistry. In this chapter, several taste sensations found in natural products are described along with their structures. Although it is still very difficult to anticipate the taste quality and intensity from the structure of an organic compound, it is expected that recent progress in the study of receptors will contribute to a full understanding of the relationship between taste sensation and chemical structure.