Snapshots along the catalytic path of KabA, a PLP-dependent aminotransferase required for kanosamine biosynthesis in Bacillus cereus UW85

• Published in: Journal of structural biology 2021-06, Vol.213 (2), p.107744-107744, Article 107744
• Main Authors: Prasertanan, Theerawat, Palmer, David R.J., Sanders, David A.R.
• Format: Article
• Language: English
• Subjects: Aminotransferase; Bacillus cereus - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Binding Sites; Catalysis; Catalytic Domain; Coenzymes - metabolism; Crystallography, X-Ray; Glucosamine - biosynthesis; Glutarates - chemistry; Glutarates - metabolism; Kanosamine; Lysine - metabolism; Models, Molecular; Protein Conformation; Protein structure; Pyridoxal Phosphate - metabolism; Transaminases - chemistry; Transaminases - genetics; Transaminases - isolation & purification; Transaminases - metabolism; X-ray crystallography
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2021.107744

[Display omitted] •KabA is a transaminase required for biosynthesis of the antibiotic kanosamine.•High resolution structures of PLP and PMP bound forms.•Structure of KabA bound with kanosamine-6-phosphate.•Reduced PLP-KabA structure with bound glutarate mimics glutamine-bound complex.•Complete structural picture of the KabA catalytic cycle. Kanosamine is an antibiotic and antifungal monosaccharide. The kanosamine biosynthetic pathway from glucose 6-phosphate in Bacillus cereus UW85 was recently reported, and the functions of each of the three enzymes in the pathway, KabA, KabB and KabC, were demonstrated. KabA, a member of a subclass of the VIβ family of PLP-dependent aminotransferases, catalyzes the second step in the pathway, generating kanosamine 6-phosphate (K6P) using l-glutamate as the amino-donor. KabA catalysis was shown to be extremely efficient, with a second-order rate constant with respect to K6P transamination of over 107 M−1s−1. Here we report the high-resolution structure of KabA in both the PLP- and PMP-bound forms. In addition, co-crystallization with K6P allowed the structure of KabA in complex with the covalent PLP-K6P adduct to be solved. Co-crystallization or soaking with glutamate or 2-oxoglutarate did not result in crystals with either substrate/product. Reduction of the PLP-KabA complex with sodium cyanoborohydride gave an inactivated enzyme, and crystals of the reduced KabA were soaked with the l-glutamate analog glutarate to mimic the KabA-PLP-l-glutamate complex. Together these four structures give a complete picture of how the active site of KabA recognizes substrates for each half-reaction. The KabA structure is discussed in the context of homologous aminotransferases.