the superfamily of 
 cytochrome p450 enzymes ( cyps ) is one of the 
 most versatile biocatalyst systems in nature .
cyps are capable 
 of catalyzing more than 20 distinct types of reactions including regio- 
 and stereoselective oxidation of unactivated c
 h bonds ( hydroxylation 
 and epoxidation ) , dealkylation , c  c bond cleavage , aromatic 
 coupling , and others . recently ,
more novel activities 
 such as decarboxylation , nitration , farnesene synthase activity , and carbene transfer have been 
 reported , reflecting the ability to further diversify the function 
 of these versatile enzymes .
catalytic activities of the vast 
 majority of cyps require one or 
 more redox partner proteins to sequentially deliver two electrons 
 from nad(p)h to the heme iron reactive center for dioxygen activation .
classically , there are two major redox partner systems , including ( 1 ) a two - component system for most bacterial and mitochondrial 
 cyps comprising a small iron  sulfur redoxin and an fad - containing 
 redoxin reductase and ( 2 ) a single fad / fmn - containing cyp reductase 
 that serves in eukaryotic microsomal p450s as a separate partner protein 
 or in a small number of bacterial cyps ( e.g. , p450bm3 ) 
 as a fused functional domain .
interestingly , nonclassical redox systems 
 have been discovered continuously , among which 
 the fmn / fe2s2-containing reductase domain termed 
  rhfred  that is naturally fused with the p450 enzyme 
 from rhodococcus sp .
ncimb 9784 has been extensively studied in our laboratories . for practical purposes due to the difficulty of obtaining 
 native 
 partners , one or more surrogate redox partners , acting either in isolation 
 or as artificially fused protein complexes ,
the 
 choice of surrogate partners or their mode of action is not necessarily 
 expected to affect the type and selectivity of reactions catalyzed 
 by p450s .
although alternative redox partners may influence catalytic 
 efficiency and/or product distribution , the chemical identity of products themselves is apparently determined 
 by the p450 enzyme .
our previous experience constructing and characterizing 
 various self - sufficient biosynthetic p450 enzymes ( whose activity 
 is independent of isolated redox partners ) fused with the rhfred reductase 
 domain also supports this  postulate  .
notably , previous work revealed that cytochrome b5 is able to modulate the bifunctional role of human cyp17a1 
 as a 17-hydroxylase or a 17,20-lyase in steroid biosynthesis .
this well - studied example demonstrates that 
 the catalytic type of a p450 enzyme can be altered by a third - party 
 protein effector via alternative interactions among the p450 enzyme , 
 p450 reductase , effector protein , and distinct substrates .
however , there have been no reports for a redoxin protein or a p450 
 reductase acting as the specific effector by itself to endow the serving 
 p450 enzyme with new catalytic activity . in this work ,
novel 
 demethylated mycinamicin products were generated 
 by p450 mycg supported by a stand - alone form of the rhfred reductase 
 domain , as opposed to rhfred fused to mycg , thus challenging the generally 
 accepted postulate .
further engineering of rhfred led to didemethylated 
 mycinamicin macrolides , demonstrating a novel route for structural 
 diversification of natural products .
protein interactions with the 
 redox partner may serve as a modulator of p450 function by dramatically 
 affecting the substrate binding mode in the p450 active site .
mycg is the multifunctional p450 monooxygenase involved in the 
 biosynthetic pathway of 16-membered ring macrolide mycinamicins in 
 the rare actinomycete micromonospora griseorubida .
physiologically , 
 it catalyzes the c14 hydroxylation , and the c12=c13 epoxidation 
 of mycinamicin iv ( m - iv ) leading to mycinamicin v ( m - v ) and mycinamicin 
 i ( m - i ) , respectively .
m - v is subsequently epoxidized by mycg , giving 
 rise to the major final product mycinamicin ii ( m - ii ) , while m - i can not 
 be further modified by mycg , thus representing the other terminal 
 product in the pathway ( figure 1a ) .
all diglycosylated 
 mycinamicins display strong antibiotic activity against gram - positive 
 bacteria and mycoplasma , and m - ii has 
 been developed into a veterinary anti - infective agent .
the multifunctional activities of mycg 
 were previously reconstituted in vitro by using the 
 commercial surrogate redox partners 
 spinach ferredoxin ( fdx ) and spinach ferredoxin - nadp reductase 
 ( fdr ) , since the native redox system driving catalytic activity of 
 mycg remains unknown .
motivated by our 
 recent work and that of others in enhancing catalytic activity 
 by fusing the rhfred reductase domain to the c - terminus of various 
 biosynthetic p450s , we constructed the mycg - rhfred fusion protein . 

similar to mycg supported by separate spinach fdx / fdr , this single - component 
 self - sufficient p450 system driven by nadph achieved all physiological 
 reactions known for mycg ( figure 1b ) .
oxidation and 
 demethylation of m - iv catalyzed by mycg using different 
 redox systems ( 2 h reactions ) .
( b ) left panel : lc traces at 280 nm of different 
 reaction extracts .
the redox systems used are shown at the left side 
 of the corresponding traces .
m - v and m - ii were coeluted , and 
 dme - m - iv and m - i were coeluted .
since m - i and m - ii lacking of the 
 diene moiety only have weak absorbance at 280 nm , the formation of 
 products appears not to be proportional to substrate consumption . 

right panel : ms / ms analysis of each mycinamicin compound ( see figure s1 for explanations of the secondary mass 
 spectra ) . ( c )
comparison of the h nmr spectra ( see figures s2 and s3 for full spectra ) of m - iv and 
 dme - m - iv
. the chemical shift and integrated peak area of the n - monomethyl group in dme - m - iv are apparently different 
 from those of the n - dimethyl group in m - iv . to investigate
if the free form 
 of the rhfred domain can support 
 the catalytic function of mycg , the ability of this two - component 
 system to convert mycinamicin iv ( m - iv ) was tested .
surprisingly , 
 in addition to the expected oxidative products , m - i , m - ii , and m - v 
 ( coeluted with m - ii ) , a new product formed with 18.5% isolated yield 
 ( table 1 ) with the same retention time as m - i 
 was observed in lc - ms analysis of the reaction mixture ( figure 1b ) .
because of its strong absorbance at 280 nm , 
 resembling that of m - iv and m - v , but distinct from m - i , we assumed 
 that the chemical structure of the new compound likely retained an 
 intact diene moiety .
indeed , its m / z value of 682.3 is 14 amu less than that of protonated 
 m - iv , suggesting a demethylated product , which was also supported 
 by high - resolution mass spectrometry ( [ m + h ] : obsd , 682.4161 ; calcd , 682.4160 ) . furthermore , 

the ms / ms spectrum of the demethylated m - iv n - demethylated 
 mycinamicin iv ( dme - m - iv ) showed two fragmentation ions of 144.2 and 
 102.2 , consistent with a demethylated desosamine moiety , while the 
 corresponding fragmentation ions for intact desosamine are 158.2 and 
 116.1 ( figures 1b and s1 ) .
detection of formaldehyde in the reaction 
 by purpald reagent provides additional 
 evidence for oxidative demethylation ( see supporting 
 information ) .
to confirm the site of demethylation , the h nmr of enzymatically prepared dme - m - iv was analyzed using 
 m - iv as reference ( figures 1c , s2 and s3 ) .
as expected , the 2.35 ppm singlet 
 peak of m - iv that corresponds to the n - dimethyl group 
 disappeared in the spectrum of dme - m - iv . instead
, a new singlet peak 
 was observed with half the integrated area at 2.72 ppm , indicating 
 the nhme group resulted from the mycg - catalyzed demethylation .
numbers of relative 
 auc280  nm ( areas under curve at 280 nm ) were calculated 
 from the integrated 
 area of certain peaks on hplc traces coupling efficiencies were calculated 
 as the percentage of nadph used for product formation over the total 
 nadph consumption this observation 
 is intriguing because a heretofore unknown dme - m - iv 
 product was obtained following separation of the previously fused 
 protein domains , mycg and rhfred .
next , we measured the oxidation 
 and demethylation activities of mycg under a variety of molar ratios 
 ( 1:1 , 1:10 , and 1:100 ) between mycg and rhfred . in all cases ,
both 
 oxidative and demethylated products were detected ( figure s4 ) , indicating the ability of mycg to catalyze demethylation 
 is not the consequence of a stoichiometric effect of rhfred .
a time 
 course of the reaction ( figure s5 ) showed 
 proportional accumulation of oxidative and demethylated mycinamicin 
 products .
sequence analysis shows that rhfred consists of two 
 functional 
 domains , the fmn domain and the fe2s2 domain . to analyze the impact of each domain on demethylation by mycg , the 
 fe2s2 domain of rhfred , which is presumably 
 involved in direct interaction with the p450 enzyme ,
was exchanged 
 for the spinach fdx , yielding a hybrid rhodococcus - spinach reductase , rhfred - fdx . when partnered with this hybrid reductase , 

the mycg - catalyzed reaction produced even greater amounts of dme - m - iv 
 ( 24.0% isolated yield , see table 1 ) , while 
 the oxidized products m - i , m - v , and m - ii were also detectable ( figure 1b ) .
this is in contrast to the mycg - rhfred - fdx fusion 
 system which , similarly to mycg - rhfred , generated only oxidized products , 
 albeit in lower quantities .
these results suggest that mycg - mediated 
 demethylation is mainly determined by the mode of interaction between 
 mycg and its redox partner rather than by the redox partner itself .
quantitatively , by fitting the substrate consumption data into 
 the one phase exponential decay curve , the rate constants ( k ) of the four studied mycg catalytic systems were determined 
 ( table 1 and figure 2a ) . in comparison , isolated rhfred and
the m - iv oxidation 
 mediated by mycg - rhfred and mycg - rhfred - fdx demonstrated the apparent 
 reaction velocity at 500 m of m - iv being 26.0  6.0 m 
 min and 8.5  2.0 m min ( k  [ s ] ) , respectively .
the steady - state 
 michaelis  menten kinetics of mycg could not be ascertained 
 due to multiple reactions occurring simultaneously .
( a ) enzymatic consumption 
 of m - iv ( calculated from hplc areas under 
 curve , auc ) fit into the one phase exponential decay curve .
( b ) the effects of catalase ( 20 u ) , superoxide dismutase 
 ( 2 u ) , ascorbate ( 10 mm ) , and the combination of these three ros scavengers 
 on the activity of mycg / rhfred against m - iv .
solid bar is the ratio 
 of demethylated product / oxidized products relative to that of the 
 control reaction without addition of any scavengers , and open bar 
 is the overall conversion percentage calculated based on the substrate 
 m - iv consumption .
it is well - known that the use of an unnatural redox partner 
 in 
 a cyp catalyzed reaction likely uncouples the electron generation 
 upon nad(p)h consumption and p450 product formation .
the uncoupling process 
 generates reactive oxygen species ( ros ) such as superoxide anions 
 ( o2 ) and h2o2 , 
 which could direct the p450 catalytic route into the  peroxide 
 shunt pathway  . to examine whether the demethylation 
 of mycinamicins is a consequence of the peroxygenase activity by undergoing 
 this shunt pathway , we first determined the nadph coupling efficiency 
 of the four catalytic systems using m - iv as substrate . as shown in 
 table 1 , the coupling efficiencies for p450-reductase 
 fusions ( 18.7% for mycg - rhfred and 21.3% for mycg
- rhfred - fdx ) were 
 lower than those of corresponding reactions using separated reductases 
 ( 29.4% for mycg + rhfred and 24.2% for mycg + rhfred - fdx ) .
these data 
 indicate that separation of reductase and p450 domains did not result 
 in greater uncoupling in these p450 catalytic systems .
moreover , 
 catalase , superoxide dismutase ( sod ) , ascorbic acid , 
 and the combination of these three ros scavengers were added into 
 the mycg / rhfred / m - iv reaction to remove ros possibly generated from 
 the uncoupling process . although these ros scavengers lowered the 
 m - iv conversion ( except for ascorbate ) as well as the ratio of demethylated 
 product to oxidized products ( except for sod ) in the majority of reactions 
 ( figure 2b ) , these effects were not dose dependent , 
 and the n - demethylation activity remained even at 
 the highest concentration of catalase , sod , and ascorbate ( figure s6 ) .
these results , together with 
 the small change of coupling efficiency 
 upon reductase domain separation , suggest that dme - m - iv is unlikely 
 to be a product of the peroxide shunt pathway .
however , the lowered 
 demethylated product / oxidized product ratio implies superoxide species 
 might still contribute to some of the observed demethylation .
this 
 could be supported by detection of a low level of h2o2 in mycg reactions ( figure s7 ) 
 and the fact that addition of ascorbic acid improved the overall conversion 
 of m - iv ( figure 2b ) since this chemical scavenger 
 is capable of protecting the enzyme and substrate / products from radical 
 damage by superoxides .
next , we 
 comparatively tested the activity of mycg , driven by each 
 of the four different redox systems , against m - iv analogues m - i , ii , 
 iii , and v , isolated from fermentation cultures of wild - type or mutant micromonospora griseorubida .
the two fused systems displayed the typical physiological set of 
 the hydroxylation and/or epoxidation products ( figure 3 ) .
however , the reactions driven by free redox partners gave 
 rise to more diversity of demethylated products .
specifically , both 
 rhfred and rhfred - fdx efficiently supported the demethylation of m - i 
 and m - iii by mycg , generating the new products dme - m - i and dme - m - iii , 
 respectively .
m - ii and m - v were demethylated by mycg at a low level 
 driven by rhfred - fdx , but not by rhfred .
of particular interest , mycg 
 partnered by the stand - alone form of rhfred - fdx was able to further 
 remove the second n - methyl group from monodemethylated 
 dme - m - i and dme - m - iii , leading to the unique double - demethylated product 
 d2me - m - i and d2me - m - iii ( figure 3 ) , respectively . 

previously , in both the in vivo(30,45 ) and in vitro reactions that employed the surrogate 
 spinach fdx / fdr system , m - i and m - ii 
 have been shown to be the two end products of the mycinamicin biosynthetic 
 pathway , and m - iii bearing the monomethoxy sugar javose is oxidized 
 by mycg at a very low level . here , the change of the redox partners 
 and their interaction mode has led to seven novel demethylated products 
 ( figures 1 , 3 , and s8 ) , demonstrating a new route for structural 
 diversification of natural products .
lc - ms analysis of the mycg reactions using 
 m - i , m - ii , m - iii , and 
 m - v as substrates .
the lc traces of m - i and m - ii were recorded at 
 240 nm .
the lc traces of m - iii and m - v were recorded at 280 nm . in 
 the m - v reactions catalyzed by two fusion mycg enzymes ,
the accumulation 
 of m - ii ( seen at 240 nm ) that is coeluted with the unreacted m - v is 
 shown in insets .
the selected ms / ms results are displayed in insets , 
 whose colors are consistent with those of the corresponding lc peaks . 

the asterisked numbers indicate the mass for the secondary ion fragment 
 of double - demethylated mycinamicins ( figure s1 ) . the structure of m - ix ( the hydroxylated m - iii labeled by the diamond 
 symbol ) is shown in figure s8 .
in this study we show 
 for the first time the ability of the multifunctional 
 p450 monooxygenase mycg to catalyze n - demethylation 
 of various mycinamicin natural product molecules .
this novel functionality 
 of mycg occurs when the monooxygenase is partnered with either the 
 free rhodococcus reductase domain , 
 rhfred , or the rhodococcus - spinach 
 hybrid , rhfred - fdx .
in contrast , no similar demethylation activity 
 was observed with the corresponding fused redox partner systems .
the 
 minor differences in the nadph coupling efficiency upon p450-reductase 
 domain separation ( table 1 ) and dose - independent 
 effects of catalase , sod , and the chemical radical scavenger ascorbate 
 on both mycg activity and the demethylation / oxidation product distribution 
 ( figure 2b ) strongly suggest that the unnatural 
 demethylation activity is primarily due to heme - iron - oxo catalysis , 
 as opposed to the peroxide shunt pathway .
thus , this finding highlights 
 a broader role of redox partner protein  protein interactions 
 in modulating catalytic activity of the p450 enzyme .
it is well - known 
 that alternative or surrogate redox partner protein(s ) might not provide 
 the optimal supporting activity toward mismatched p450 enzymes and could influence product distribution .
the modulating effect of a third party protein , cytochrome b5 , on the hydroxylase / lyase bifunction of cyp17a1 
 supported by cytochrome p450 reductase has also been demonstrated .
however , 
 to the best of our knowledge , the entirely new catalytic activity 
 of a p450 enzyme associated with an alternative redox partner is unprecedented .
the novel catalytic activity demonstrated by mycg likely stems 
 from the poor accessibility of the substrate binding site , as evidenced 
 by the x - ray structure analysis of the mycg - substrate complexes .
thus , the bulky and conformationally restrained 
 mycinamicin substrates were largely unable to penetrate the l - shaped 
 binding site to adopt a catalytically productive binding mode .
nmr 
 modeling using paramagnetic restraints suggests that displacement 
 of a number of the protein secondary structure elements takes place 
 in order to accommodate mycinamicin substrates in an orientation conducive 
 to the observed oxidation pattern . by 
 analogy to p450cam and the human p450 3a4 , reorganization of the mycg active site
may be 
 driven by binding of the redox partner . as has been recently demonstrated 
 by elegant work from the poulos laboratory , binding of the endogenous redox partner putidaredoxin
stabilizes 
 the  open  conformation of p450cam , facilitating substrate 
 access to the active site .
consistent with the structurally 
 resolved mycg - substrate complexes , mycinamicins 
 exhibiting pseudo two - fold symmetry 
 enter the active site in one of the two possible orientations :  mycinose - in - desosamine - out  
 ( figure 4a ) or  desosamine - in - mycinose - out  
 ( figure 4b ) .
the desosamine - in - mycinose - out 
 entry results in the catalytically nonproductive binding mode represented 
 by the m - iii costructure ( pdb i d code 2yca ) . remarkably , the mycinose - in - desosamine - out 
 entry in the absence of the redox partner resulted in both the m - iv 
 and m - v substrates being stalled in the intermediate nonproductive 
 binding pose in all the structurally resolved complexes .
if substrate relocation to the catalytically 
 competent mode is indeed initiated by specific interactions with the 
 endogenous redox partner , the alternative surrogates may to some extent 
 mimic this allosteric effect , either facilitating or hampering substrate 
 progression to the catalytic site .
tethered to mycg , both rhfred and 
 rhfred - fdx afforded physiological product profiles , with the qualification 
 that the product yield was low for mycg - rhfred - fdx . alternatively , 
 when used as stand - alone redox partners , both the rhfred and rhfred - fdx 
 domains produced demethylated ( rhfred ) or didemethylated ( rhfred - fdx ) 
 products of not only the native mycg substrates m - iv and m - v but also 
 the m - iii precursor , an otherwise poor substrate for mycg oxidation , 
 which was efficiently demethylated ( figure 3 ) .
( a ) a putative binding pose of m - iv leading to physiological 
 c14 hydroxylation or / and c12c13 epoxidation , which might be 
 derived from the noncatalytic  mycinose - in - desosamine - out  
 entry pose as observed in the 2y98 ( pdb i d ) structure .
( b ) a putative  desosamine - in - mycinose / javose - out  
 binding pose yielding n - demethylation is likely to 
 be induced or stabilized by protein 
the noncatalytic conformation observed 
 in the 2yca structure 
 may be derived from this pose .
slice through the mycg binding site shows m - iv or m - iii in virtually 
 orthogonal orientations experimentally observed in the crystal structures .
protein surface is colored by hydrophobicity , 
 hydrophobic areas are in orange , and hydrophilic areas in blue .
to explain this phenomenon we 
 speculate that while the electron 
 transfer between mycg and the surrogate redox partners is retained , 
 the allosteric effect facilitating substrate progression to the normal 
 catalytically competent binding mode (  mycinose - in - desosamine - out  ) 
 is likely diminished , if not entirely lost .
this might be due to intrinsically 
 low affinity between the surrogate partners and mycg , particularly 
 if concentration dependence in binary complex formation becomes a 
 rate - limiting issue in the bimolecular reaction .
also , loss of the 
 positioning effect of the linker following domain separation may be 
 a factor in changing the interaction mode of free reductase and p450 
 domains .
we conclude that in the absence of the allosteric influence 
 of the redox partner , mycinamicins are more inclined to be stalled 
 in the  desosamine - in - mycinose - out  noncatalytic entry 
 pose , increasing the probability of n - demethylation 
 occurring in an otherwise atypical reaction site . despite discovery 
 of a new n - demethylation activity 
 for monooxygenase mycg , caution
should be exercised in interpreting 
 reconstituted p450 activity generated in a surrogate redox system , 
 particularly until the generality of this approach is further investigated . 

however , an interesting hypothesis is suggested : p450 enzymes could 
 be even more versatile in vivo than previously considered , 
 because these biocatalysts might interact with a variety of redox 
 partners to gain alternative activities .
this may impart evolutionary 
 advantages to the host organisms through detoxifying a more diverse 
 range of xenobiotics or synthesizing more secondary metabolites to 
 adapt to ever - changing environments .
clear indications that the p450-omes 
 of microbes ( e.g. , streptomyces coelicolor ) are capable of interacting with a limited 
 set of redox partners are consistent with a broader role for redoxin - mediated 
 selectivity in these fascinating enzyme systems .
 cytochrome 
 p450 enzymes are capable of catalyzing a great variety 
 of synthetically useful reactions such as selective c  h functionalization . 
 
 surrogate redox partners are widely used for reconstitution of p450 
 activity based on the assumption that the choice of these auxiliary 
 proteins or their mode of action does not affect the type and selectivity 
 of reactions catalyzed by p450s . herein , we present an exceptional 
 example to challenge this postulate . 
 mycg , a multifunctional biosynthetic 
 p450 monooxygenase responsible for hydroxylation and epoxidation of 
 16-membered ring macrolide mycinamicins , is shown to catalyze the 
 unnatural n - demethylation(s ) of a range of mycinamicin 
 substrates when partnered with the free rhodococcus reductase domain rhfred or the engineered rhodococcus - spinach hybrid reductase rhfred - fdx . 
 by contrast , mycg fused with 
 the rhfred or rhfred - fdx reductase domain mediates only physiological 
 oxidations . 
 this finding highlights the larger potential role of variant 
 redox partner protein  
 protein interactions in modulating the 
 catalytic activity of p450 enzymes . 
Introduction
Results
Discussion