Structural basis of the correct subunit assembly, aggregation, and intracellular degradation of nylon hydrolase

Abstract

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a β subunit (9 kDa). Four αβ heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

X‐ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase

“…An L137A substitution in NylC p2 resulted in complete loss of autocleavage (Table 1) [11]. By contrast, the L137A mutation in the thermostable D122G mutant resulted in partial loss of autocleavage, and the conversion was barely affected even after incubation at 37 °C (cleavage ratio = 33–34%) (Fig.…”

“…The plasmids pSKFC4 (NylC p2 ) and pSKRC4 (NylC A ) contained 1.1 kb genes flanked by BamHI and PstI restriction sites, which were cloned into the expression vector pBluescript II SK(+) (Stratagene, La Jolla, CA, USA) [8,10]. Plasmids encoding NylC p2 -G 122 , -Y 130 , −A 137 , -G 122 Y 130 , -G 122 Y 130 A 36 Q 263 and -G 122 A 137 enzymes have been cloned into pBluescript II SK(+) [8,11]. To introduce N266A, N266G, N219A, F134A, N219A and D306A single mutations and N266G/T267C double mutation into the NylC p2 sequence, site-directed mutagenesis was performed using the PrimeSTAR mutagenesis Basal kit (Takara Bio Inc., Kusatsu, Japan), pSKFC4 (template DNA) and primers listed in Table S6.…”

“… c The activity of NylC p2 ‐G 122 Y 130 A 36 Q 263 was assayed independently in references [8, 11]. Because the activities varied from 3.9 to 6.8 U·mg −1 , depending on the purification/storage conditions of the enzyme and differences in the composition of the substrate (contents of oligomers with different polymerization), the average value of the two experiments is shown. …”

Characterization and engineering of branched short-chain dicarboxylate metabolism in Pseudomonas reveals resistance to fungal 2-hydroxyparaconate

“…Ideally, polymers should be designed to be both bio-based and biodegradable to show a high degree of sustainability while also reducing the environmental burden ( Wei et al, 2020 ). In recent years, major progress was achieved in the (bio-) catalytic depolymerization of plastics including polyesters, polyurethanes, and polyamides ( Ellis et al, 2021 ; Magnin et al, 2020 ; Negoro et al, 2018 ; Tournier et al, 2020 ). Due to the heterogenic composition of many plastic products, their depolymerization results in a diverse mixture of monomeric building blocks.…”

On the Role of Platelet-Generated Amyloid Beta Peptides in Certain Amyloidosis Health Complications

“…Formation of β-pleated sheet hydrogen bonds between two or more parallel protein strands requires standard spacing between amino acids in these parallel polypeptides. It also requires correct subunit assembly and the right organization of the process ( 1 ). Unfortunately, this bond formation between parallel chains may occur pathologically because of mutations augmenting the binding propensity of particular polypeptides or the elevated concentration or overproduction of specific peptide chains, allowing the formation of polymeric ß-pleated sheets consisting mainly of multiple copies of the same type of chain.…”