Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars

Abstract

This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-specific sugar nucleotides. The latter, which include UDP-hexoses such as UDP-diNAc-bacillosamine (UDP-diNAcBac), UDP-diNAcAlt, and UDP-2,3-diNAcManA, are also important components of other bacterial and archaeal glycoconjugates. The ready availability of these "high-value" intermediates will enable courses of study into inhibitor screening, glycoconjugate biosynthesis pathway discovery, and unnatural carbohydrate incorporation toward metabolic engineering.

Cofactor‐Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides

“…Nevertheless, AcCoA is too expensive to do large‐scale synthesis. Chemical acetylation has been proven to produce acetylated sugar nucleotides in multimilligram‐scale, [44] but it needs the separation of amino sugar in pure form. Therefore, we turn to do the enzymatic acetylation by employing a AcCoA regeneration system.…”

“…The subsequent amination of UDP‐4‐keto‐6‐deoxy‐GlcNAc or UDP‐4‐keto‐6‐ deoxy‐Glc at the C‐4 position by transaminase resulted in UDP‐ D‐4n‐FucNAc or UDP‐D‐4n‐QuiNAc [43] . However, it is difficult to obtain pure UDP‐D‐4n‐FucNAc or UDP‐D‐4n‐QuiNAc in sufficient quantity by enzymatic synthesis [43, 44] . The amino donor of transaminase is PMP, which is regenerated from pyridoxal‐5′‐phosphate (PLP) and L‐Glutamate without the need of an exogenous enzyme (Figure 1, CRS4).…”

Metabolic Engineering of Corynebacterium glutamicum for Production of UDP-N-Acetylglucosamine

“…UDP-linked amino sugars are difficult to synthesize, however, chemical, enzymatic and chemoenzymatic approaches have been explored ( Koizumi et al, 1998 ; Ahmadipour and Miller 2017 ; Zamora et al, 2017 ; Ahmadipour et al, 2018 ; Ahmadipour et al, 2019 ; Mikkola, 2020 ). Chemical synthesis of activated carbohydrates is demanding and relies on specialized expertise in carbohydrate chemistry; however, due to the lack of alternative routes, it remains a popular choice to synthesize non-natural sugar nucleotides (e.g., fluorinated nucleotide sugars) ( Wagner et al, 2009 ; Tedaldi et al, 2012 ), which may serve for the synthesis of modified oligosaccharides, as enzyme inhibitors and/or in diagnostics ( Mikkola, 2020 ).…”

“…Moreover, chemical approaches also suffer from other major drawbacks such as lack of stereoselectivity, tedious purification processes, and a low space-time yield ( Zhao et al, 2010 ). Chemoenzymatic syntheses use biotransformation steps together with simple chemical steps, thus improving efficiency and enabling the synthesis of chemically defined nucleotide-activated sugars ( Zamora et al, 2017 ). Using glucosamine and ATP as substrates and hexokinase, GlmM, and GlmU, Heidlas et al (1992) firstly showed the enzymatic synthesis of UDP-GlcNAc.…”

Meeting Proceedings from ICBS 2018—Toward Translational Impact

“…21 Cristina Zamora (MIT, USA) closed the session by describing her studies of Campylobacter jejuni pathogenicity, one of the main causes of gastroenteritis. 22 Using an innovative human intestinal immune-competent microphysiological system, Zamora explored the role of N-glycans in C. jejuni infection via the Pgl pathway. She uncovered the multifaceted importance of the Pgl pathway in C. jejuni host−pathogen interactions and characterized the importance of chemokine and cytokines profiles and the different composition of virulence-associated outer-membrane vesicles in pathogenesis.…”