Catalytic hydroxylation of phenylcyclohexane 1 by wild-type and the Y96A and Y96F mutant forms of cytochrome P450cam occurs only at the 3-and 4-positions on the cyclohexane ring, giving cis-3-phenylcyclohexanol 2, cis-4-phenylcyclohexanol 3 and trans-4-phenylcyclohexanol 4.

A highly stereo‐ and regioselective linkage for the β‐1,4‐mannoside bond of trisaccharide 1 (R/R′ = H/OH) is achieved with a redesigned recombinant biocatalyst: a suitably modified yeast enzyme (ALG1) was expressed in E. coli; the enzyme was then immobilized on a NiII affinity column.

Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-3H]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [3H]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.

Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of glycosyltransferases. The role of dolichol in enzyme substrate recognition is investigated. Thus the biosynthetic intermediate NN'-diacetylchitobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as substrates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[3H]Mannosylated lipids from this reaction were then used as substrates for the subsequent mannosyltransferases from yeast or rat liver microsomes. It was found that both the dolichyl- and phytanyl-linked substrates were easily mannosylated to form Man5GlcNAc2, with some further mannosylation to Man7GlcNAc2 and Man9GlcNAc2 at low concentrations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransferases in the biosynthetic pathway for N-glycosylation.

The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1-->4GlcNAc beta 1-->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.