Peter A B Orlean

Peter A B Orlean

p-orlean@illinois.edu

B213 Chemical and Life Sciences Laboratory
601 S. Goodwin Ave.
Urbana, IL 61801
Office: (217) 333-4139

Mail to: B213 CLSL, MC-110
601 S. Goodwin Ave.
Urbana, IL 61801

Professor of Microbiology

Research Topics

Enzymology, Glycobiology, Membrane Biology

Education

B.Sc. (Bacteriology), University of Newcastle-upon-Tyne, 1977
M.Phil. (Biochemistry), University of Cambridge, 1978
Ph.D. (Biochemistry), University of Cambridge, 1982
Post-Doctoral (Cell Biology), Universität Regensburg, 1983-1985
Post-Doctoral (Molecular Biology), Massachusetts Institute of Technology, 1985-1990

Teaching Interests

Mechanism and regulation of polysaccharide synthases; cell wall biogenesis in yeast and pathogenic fungi

We study how insoluble, structural polysaccharides are made from soluble monomers, focusing on chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), which is a signature structural component of fungal cell walls and the exoskeletons of arthropods.

Chitin synthases (CSs) are multispanning membrane proteins with a cytoplasmic domain that catalyzes transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to elongate chitin chains, which are concomitantly extruded across the plasma membrane through the enzyme’s translocation channel. We are exploring how polymerization is initiated de novo, whether a primer molecule is involved, and what factors determine the length of enzymatically synthesized chitin chains. Key findings and their implications are as follows:

Chitin chains can be initiated on monomeric primers. Using a combination of biochemistry, chemical biology, and mass spectrometry, we have shown that the free chitin monomer, GlcNAc, as well as the GlcN analogues N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine, all prime formation of chitin oligosaccharides and insoluble chitin by a yeast CS (Figure 1). Our analyses of chitin oligosaccharide synthesis also provided the first direct evidence that chitin synthases can add monomers one sugar at a time.

Development of a novel strategy to estimate average chitin chain length. We have shown that free GlcNAc is incorporated into insoluble chitin in a manner dependent on UDP-GlcNAc. This allowed us to develop a novel approach to estimating the average chain length of in vitro-synthesized chitin, in which chain length is determined from the ratio of moles of UDP-GlcNAc-derived, chain elongating GlcNAc to moles priming GlcNAc incorporated at one end of the chitin chain (Figure 2). Chain lengths estimated by our new ratio method are dependent on two variables: rate of [UDP-GlcNAc]-dependent elongation and rate of [GlcNAc]-dependent initiation. Our method yields a combined parameter that describes the activities of CSs in terms of relative abilities to initiate on a priming GlcNAc and then to extend the glycan, and represents a new theoretical approach to describing polysaccharide synthase activity.

New proposals for chitin synthase mechanism. Consideration of our experimental findings in the context of the current the structure-based catalysis-translocation model for processive polysaccharide synthesis leads us to make the following proposals:

1. CSs may “self-prime” by hydrolyzing UDP-GlcNAc. Although free GlcNAc primes chitin synthesis in vitro, it is not formed biosynthetically in vivo. However, a CS could generate its own priming GlcNAc by catalyzing hydrolysis of UDP-GlcNAc instead of glycosyltransfer, and then extend that GlcNAc into a chitin chain (Figure 3).

2. CSs may synthesize chains in bursts. Our finding that free GlcNAc and acylamido GlcN analogs prime formation of insoluble chitin implies that a CS’s active site is not constantly occupied by the end of a preexisting, growing chitin chain, rather, that the active site can be empty at times and can accommodate a monomeric primer, allowing de novo chain priming. Therefore, CSs may make polysaccharide chains in bursts, release them, and then reinitiate chitin synthesis on a primer (Figure 3).

Next steps.

1. Do CSs have UDP-GlcNAc hydrolytic activity? This could be explored using a UDP-GlcNAc derivative that releases a diagnostic GlcNAc analogue that cannot itself act as a primer.

2. Do different chitin synthases make different chain length distributions? Chitin-producing organisms typically have two, often several more CSs, and these may make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. How does chitin chain length impact fungal cell wall construction?

3. Can chitin synthases make novel polymers using UDP-derivatives of 2-acylamido GlcN? It remains to be established whether CSs can use UDP-linked derivatives modified at the 2-position of the sugar, for example, UDP-N-butanoylglucosamine. The possibility of introducing side chain modifications during chitin biosynthesis using UDP-GlcNAc analogs could be exploited to generate polymers with novel properties, or ones bearing groups that could be derivatized post-synthetically.

Awards

American Cancer Society Junior Faculty Research Award, 1993-1996
Helen Corley Petit Professorship, UIUC College of Liberal Arts and Sciences, 1997-1998
University Scholar, University of Illinois, Urbana, 1997-1998
Burroughs Wellcome Scholar Award in Molecular Pathogenic Mycology, 1998-2005
Visiting Professor, Kungliga Tekniska Högskolan, Stockholm, Sweden, Division of Glycoscience 2013

Representative Publications

Orlean, P. and Funai, D. (2019) Priming and elongation of chitin chains: implications for chitin synthase mechanism. The Cell Surface, Vol. 5, Dec. 2019, in press. https://doi.org/10.1016/j.tcsw.2018.100017

Gyore, J., Parameswar, A.R., Hebbard, C., Oh, Y., Bi, E., Demchenko, A.V., Price, N.P., and Orlean, P. (2014) 2-Acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2. Journal of Biological Chemistry 289: 12835-12841.

Zhang, Y., Askim, J.R., Zhong, W., Orlean, P., and Suslick, K.S. (2014) Identification of pathogenic fungi with an optoelectronic nose. Analyst 139: 1922-1928.

Orlean, P. (2012) Architecture and Biosynthesis of the Yeast Cell Wall. In: YeastBook. Cell Signaling & Development (P. Pryciak and J. Thorner, Eds.) Genetics, 192: 775-818.

Oh, Y., Chang, K.-J., Orlean, P., Wloka, C., Deshaies, R., and Bi, E. (2012) Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast. Mol. Biol. Cell 23: 2445-2456.

Scarcelli, J.J., Colussi, P.A., Fabre, A.-L., Keller, M., Boles, E., Orlean, P., and Taron, C.H. (2012) Uptake of radiolabeled GlcNAc into Saccharomyces cerevisiae via native hexose transporters and its in vivo incorporation into GPI precursors in cells expressing heterologous GlcNAc kinase. FEMS Yeast Research 12: 305-316.

Complete Publications List