Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Prior research has established the general role of hydroxylase enzymes in catalyzing hydroxylation reactions in aromatic compounds. However, the precise amino acid sequence of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens remained unclear. Earlier studies focused on enzyme function and crystal structures of related proteins, but did not resolve the full sequence of this specific monomer. This gap motivated the integration of X-ray crystallographic data with protein sequencing techniques to clarify the monomer’s structure. No prior work had resolved the exact amino acid composition of this hydroxylase. The lack of detailed sequence information limited further functional studies. Researchers needed a complete sequence to understand the enzyme’s catalytic mechanism and evolutionary relationships. This paper addresses that need by combining two distinct analytical approaches.

The aim of this work was to determine the amino acid sequence of the p-hydroxybenzoate hydroxylase monomer from Pseudomonas fluorescens. The enzyme plays a role in the hydroxylation of aromatic substrates, but its sequence had not been fully characterized. By integrating X-ray crystallography data with protein sequencing methods, the researchers sought to resolve the monomer’s structure in detail. This approach allowed for a more accurate determination of the sequence than either method could achieve alone. The study focused on a specific monomer within a larger enzyme complex. The goal was to provide a complete amino acid sequence for further biochemical and structural investigations. This information is necessary for understanding the enzyme’s catalytic properties and potential applications. The findings may also inform studies on related hydroxylases in other bacterial species.

The researchers combined X-ray crystallographic data with direct protein sequencing to determine the amino acid sequence of the monomer. The crystallographic study was conducted at a resolution of 0.25 nm, providing structural details of the enzyme. Protein sequencing was performed using standard biochemical techniques to identify the amino acid composition. The monomer was isolated from the enzyme complex for analysis. The sequence was reconstructed by aligning the crystallographic data with the sequencing results. This dual approach allowed for a more accurate determination of the sequence than either method alone. The monomer’s polypeptide chain was analyzed in its entirety. The final sequence was validated by comparing it with known structural motifs and enzyme families.

The monomer consists of a single polypeptide chain containing 394 amino acids. The molecular weight of the monomer was calculated to be 44,299. The sequence was determined by integrating X-ray crystallography and protein sequencing data. The crystallographic study provided detailed structural information at 0.25 nm resolution. The amino acid sequence was validated by comparing it with known structural motifs. The monomer’s sequence is now fully characterized for the first time. This finding provides a foundation for future studies on the enzyme’s structure-function relationship. The results may also help in comparing this hydroxylase with other similar enzymes in related bacterial species.

The study successfully determined the amino acid sequence of the p-hydroxybenzoate hydroxylase monomer from Pseudomonas fluorescens. The sequence was derived from a combination of X-ray crystallography and protein sequencing methods. The monomer contains 394 amino acids and has a molecular weight of 44,299. This finding fills a knowledge gap in the structural characterization of this enzyme. The sequence provides a basis for further biochemical and structural investigations. The integration of crystallographic and sequencing data proved effective for this purpose. The results may support comparative studies with other hydroxylases in related bacteria. The authors suggest that this information could be useful for future functional and evolutionary analyses.