Three novel mutations in α-galactosidase gene involving in galactomannan degradation in endosperm of curd coconut.

The deficiency of α-galactosidase activity in coconut endosperm has been reported to cause a disability to hydrolyze oligogalactomannan in endosperm resulting in curd coconut phenotype. However, neither the α-galactosidase encoding gene in coconut nor the mutation type has been identified and characterized in normal and curd coconuts. In this study, cDNA and genomic DNA encoding α-galactosidase gene alleles from a normal and two curd coconuts were successfully cloned and characterized. The deduced amino acid of wild type α-galactosidase contains 398 amino acid residues with a 17 N-terminal amino acids signal peptide sequence. Three mutant alleles, the first 19-amino acids from 67 to 85 (ADALVSTGLARLGYQYVNL) deletion with S137R and the second R216T, were identified from curd coconut plant no.1 while the third P250R was identified from curd coconut plant no. 10. All mutations of α-galactosidase gene were confirmed by the analysis of parental genomic DNA from normal and curd coconuts. Heterologous expression in Komagataella phaffii (Pichia pastoris) indicated that recombinant P250R, R216T and 19-amino acids deletion-S137R mutant proteins showed no α-galactosidase activity. Only the recombinant wild-type protein was able to detect for α-galactosidase activity. These results are in accordance with the no detection of α-galactosidase activity in developing curd coconut endosperms by tissue staining. While, the accumulation of enzyme activity was present in the solid endosperm of normal coconut. The full-length cDNA and parental genomic DNA sequences encoding α-galactosidase in normal coconut as well as identified curd coconut mutant alleles are reported in Genbank accession no. KJ957156 and KM001681-3. Transcription level of the α-galactosidase gene in mature curd coconut endosperm was at least 20 times higher than normal. In conclusion, absence of α-galactosidase activity caused by gene mutations associates with an accumulation of oligogalactomannan in endosperms, resulting in curd coconut phenotype.

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain.

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ∼20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.

Ultrafast solvation dynamics of flavin mononucleotide in the reductase component of p-hydroxyphenylacetate hydroxylase.

The reductase unit of p-hydroxyphenylacetate hydroxylase contains flavin mononucleotide (FMN) as a cofactor. Fluorescence decay curves measured by fluorescence up-conversion method were remarkably dependent on monitored emission wavelength. The fluorescence lifetime was shorter at the shorter emission wavelengths and longer at the longer wavelengths. Spectral shift correlation function of p-coumaric acid in water and FMN in C1 protein in buffer solution were expressed by two-exponential functions. Correlation times, phi1 and phi2, of p-coumaric acid were 0.053 and 0.650 ps, respectively, which was similar to previous works. phi1 and phi2 of C1 were 0.455 and 250 ps, respectively. The Stokes shift from t=0 to t=infinity was 2200 cm(-1), while it is 500 cm(-1) in the static Stokes shift obtained by the solvent effect of the fluorescence spectrum under static excitation. This suggests that the isoalloxazine ring of FMN in C1 is exposed in hydrophilic environment. Such large Stokes shift was unusual among flavoproteins. The biphasic decay of the spectral correlation function in C1 was discussed and compared to the biphasic decay of tryptophan in proteins.

Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase.

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C1) and an oxygenase (C2). C1 catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C2 in the hydroxylation reaction. C1 is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C1-bound FMN can be reduced and released to be used efficiently as the substrate for the C2 reaction; additional FMN is not necessary. Reactions in which O2 is rapidly mixed with solutions containing C1-FMNH- and C2 are very similar to those in which solutions containing O2 are mixed with one containing the C2-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C2 and has already been completely transferred to C2 before it reacts with oxygen. Rate constants for the transfer of FMNH- from C1 to C2 were found to be 0.35 and >or=74 s-1 in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C1. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s-1, while with HPA present it dissociates at 80 s-1; these are the same rates as those for the transfer from C1 to C2. Therefore, the dissociation of FMNH- from C1 is rate-limiting in the intermolecular transfer of FMNH- from C1 to C2, and this process is regulated by the presence of HPA. This regulation avoids the production of H2O2 in the absence of HPA. Our findings indicate that no protein-protein interactions between C1 and C2 are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C1.

Transglucosylation of tertiary alcohols using cassava beta-glucosidase.

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.