Cloning and <i>in silico</i> characterization of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from <i>Bacillus subtilis</i>

Quang Huy Nguyen; Thi Thanh Xuan Nguyen; Minh Thu Nguyen; Luong Luan Chu

doi:10.15625/vjbt-23418

Author affiliations

Authors

Quang Huy Nguyen \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam https://orcid.org/0000-0001-7394-6209
Thi Thanh Xuan Nguyen \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Nghia Do, Hanoi, Vietnam
Minh Thu Nguyen \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam
Luong Luan Chu \(^1\) National Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam https://orcid.org/0000-0001-9223-6798

DOI:

https://doi.org/10.15625/vjbt-23418

Keywords:

3-hexulose-6-phosphate synthase, 6-phospho-3-hexuloseisomerase, hxlA, hxlB, Bacillus subtilis ST123

Abstract

3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are key enzymes of the ribulose monophosphate (RuMP) pathway, which plays an essential role in formaldehyde assimilation and detoxification in methylotrophic microorganisms. Although homologous genes encoding these enzymes are annotated in the genome of Bacillus subtilis, their molecular and structural characteristics remain poorly explored in this organism. In this study, taxonomic identification using Kraken2, 16S rRNA phylogenetic analysis, and multilocus sequence typing confirmed the strain as B. subtilis ST123. Moreover, the hxlA and hxlB genes encoding HPS and PHI were cloned from a local B. subtilis ST123 strain isolated from infant fecal samples. The amplified hxlA (633 bp) and hxlB (558 bp) genes were successfully cloned, sequenced, and analyzed. Sequence alignment revealed high conservation with reference B. subtilis sequences. The three-dimensional structure of HPS was predicted using AlphaFold3 and rigorously evaluated by MolProbity, ERRAT, Verify3D, and ProSa. The HPS model exhibited high overall structural quality, with a MolProbity score of 1.13, 100% residues in the Ramachandran favored region, and an ERRAT quality factor of 99.505, although localized regions of lower sequence-structure compatibility were identified by Verify3D. The PHI structure was analyzed based on an available X-ray crystal structure (PDB ID: 1M3S). Physicochemical characterization indicated that HPS is thermostable and suitable for heterologous expression, whereas PHI showed a higher instability index, suggesting potential challenges during in vitro expression. This work provides the first comprehensive cloning and in silico characterization of HPS and PHI from a Vietnamese B. subtilis strain, offering valuable insights for metabolic engineering, synthetic biology, and C1 assimilation pathway design.

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