No abstract

N-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn 2+ at 1.5 and 1.8 A Ê resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining b-glycosidases. The structuring of a 13 residue loop, resulting from UDPGlcNAc/Mn 2+ binding, provides an explanation for the ordered sequential`Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine b-1,4-galactosyltransferase 1 and Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain de®ne a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 Å resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA′/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

A key enzyme in regulating the maturation of N-linked glycans is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC 2.4.1.101). Lec1 CHO cells lack GlcNAc-TI activity and synthesize only the oligomannosyl class of N-glycans. By contrast, Lec1A CHO mutants have weak GlcNAc-TI activity due to the reduced affinity of GlcNAc-TI for both the UDP-GlcNAc and Man(5)GlcNAc(2)Asn substrates. Lec1A CHO mutants synthesize hybrid and complex N-glycans, albeit in reduced amounts compared to parental CHO cells. In this paper, we identify two point mutations that gave rise to the Lec1A phenotype in three independent Lec1A CHO mutants. The G634A mutation in Lec1A.2C converts an aspartic acid to an asparagine at amino acid 212, disrupting a conserved DXD motif (E(211)DD(213) in all GlcNAc-TIs) that makes critical interactions with bound UDP-GlcNAc and Mn(2+) ion in rabbit GlcNAc-TI. The C907T mutation in Lec1A.3E and Lec1A.5J converts an arginine conserved in all GlcNAc-TIs to a tryptophan at amino acid 303, altering interactions that are important in stabilizing a critical structural element in rabbit GlcNAc-TI. Correction of each mutation by site-directed mutagenesis restored their GlcNAc-TI activity and lectin binding properties to parental levels. The effect of the two amino acid changes on GlcNAc-TI catalysis is discussed in relation to the crystal structure of rabbit GlcNAc-TI complexed with manganese and UDP-GlcNAc.