Figure 1.
Proposed propylproline biosynthetic sub-pathway of the lincomycin (A) biosynthesis.
The sub-pathway leading to propylproline formation (B) was proposed by determining the biosynthetic origin of the carbon and nitrogen atoms using feeding studies and subsequent NMR analysis [2]. Two intermediates (indicated by asterisks) were confirmed experimentally [4], [5]. The Lmb proteins known to be involved in the sub-pathway are indicated.
Figure 2.
Spectrometric analysis of MBP2*- LmbB2.
Absorption spectra (A) of oxidized (dashed line) and reduced (full line) forms of MBP2*- LmbB2 bore witness of heme presence in the MBP2*- LmbB2 molecule showing Soret band at 404 nm and its shift under dithionite treatment. Reduced CO-differential spectrum (B) of the MBP2*- LmbB2 did not show a maximum at 450 nm indicating that LmbB2 does not belong to the cytochrome P450 superfamily.
Figure 3.
Resonance Raman spectrum of MBP2*- LmbB2.
MBP2*- LmbB2 (trace A) was superimposed on a fluorescence background. Background corrected spectrum (trace B) was expanded 25× to highlight details. Upper part of the background-corrected spectrum exhibited pattern typical for the Soret band-excited RRS spectra of various heme-containing proteins [21], [22].
Figure 4.
Dependence of the LmbB2 reaction rate on tyrosine/BH4 concentration.
Dependence of the LmbB2 reaction rate on tyrosine (A) and BH4 (B) concentration and their respective double logarithmic plots (C, D). The velocity data represent the average of three measurements; vertical error bars represent standard deviation.
Table 1.
Effect of metal chelators, detergents, electron donors on the LmbB2 activity.
Figure 5.
Analysis of LmbB2 by CD spectroscopy.
(A) The far-UV CD spectrum of LmbB2. (B) Thermal denaturation of LmbB2 in the presence of 0 µM (blue curve), 50 µM (red curve) and 250 µM (green curve) concentration of BH4 bear witness of the stabilization of the LmbB2 structure by BH4.
Table 2.
Effect of BH4 on melting temperature (Tm) of LmbB2.