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  1. Evaluation Summary:

    This manuscript describes an interesting regulation of the hexosamine biosynthetic pathway (HBP) that is relative specific to the mouse embryonic stem cells (mESC). HBP produces UDP-N-acetylglucosamine, which is used in various protein glycosylation events, thus regulating many biological pathways. Understanding this pathway and its regulation is thus of fundamental significance.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Using chemical mutagenesis to screen for mutants in mESC that are resistant to tunicamycin, the authors of this manuscript found that mutations in N-acetylglucosamine deacetylase AMDHD2 confer resistance. Furthermore, mESC expresses glutamine fructose-6-phosphate amidotransferase 2 (GFAT2) instead of GFAT1. The authors showed GFAT1 is more efficiently inhibited by UDP-GlcNAc than GFAT2. Thus, the hydrolysis of N-acetylglucosamine by AMDHD2 helps to control the level of HBP in mESC by counteracting the action of GFAT2. This can also explain why mutations in AMDHD2 could confer resistant to tunicamycin as the mutations would increase UDP-GlcNAc levels in mESC. The authors also solved the structure of AMDHD2 and showed the many of the mutations either decreased the catalytic activity, folding or protein stability. This study thus provides important new insight into the hexosamine biosynthesis pathway (HBP).

    Overall, this is a very nice study with many strengths. The chemical mutagenesis and screening for tunicamycin resistant is a nice method that allows the identification of the role of AMDHD2. The structural and biochemical characterization of AMDHD2 is beautifully done. The differential expression patterns and feedback inhibition profiles of GFAT1 and GFAT2 also provided important insights to understand their functional differences and why AMDHD2 mutations more specifically affects mESC that expresses GFAT2.

    I could not point out any major weakness with the study. If I have to be very critical, I would say that it is not clear how GFAT2 and AMDHD2 avoid forming a futile cycle in HBP. However, this is a question better suited for future studies. One very minor weakness is that the down-regulation of GFAT2 protein level during neuronal differentiation is very modest, which in contrast to the very dramatic differences in mSEC and N2a cells.

  3. Reviewer #2 (Public Review):

    The paper by Kroef et al. is aimed at identifying novel regulators of hexosamine biosynthesis - a key pathway that feeds into different types of essential glycosylation processes. The authors use a recently developed haploid stem cell line, and a tunicamycin sensitivity phenotype to perform a genetic screen for novel recessive alleles. Building on their previous work that shows how UDP-GlcNAc provides direct feedback inhibition of GFAT1, here they uncover that AMDHD2 provides another feedback loop for conditions where GFAT2 is the dominant isoform. A crystal structure of this enzyme and a discussion of its mechanism is also included. There were also attempts to identify the role of AMDHD2 using a mouse knockout. This represents a significant advance of knowledge in this area and is of considerable interest to the field and the wider eLife readership. However, the authors do not provide sufficient data to support their proposed mechanism of GFAT2/ AMDHD2 regulation of the HBP, and several important controls are missing. These issues will need to be addressed in a revised manuscript.